Targeted nanocarrier comprising modified exosome

Abstract

A targeted nanocarrier comprising a modified exosome. The modified exosome may comprise an interior core comprising an aqueous medium of the modified exosome, and at least one bilayer membrane. The bilayer membrane may comprise a plurality of phospholipid units, a plurality of amphiphilic copolymer units, and at least one assembled targeting ligand.

Description

TARGETED NANOCARRIER COMPRISING MODIFIED EXOSOME TECHNICAL FIELD

[0001] The present disclosure is generally related to an exemplary nanocarrier, and more particularly to an exemplary nanocarrier comprising an exemplary modified exosome for delivering drugs to targeted cells.BACKGROUND

[0002] Recently, extensive studies have been carried out to diagnose and treat diseases using the system of drug nanocarriers. Polymeric nanocapsules, as a type of drug carrier, have high efficiency in this field because they have the possibility of loading drugs and directly transferring them to cancer cells, thereby increasing the therapeutic effect and reducing the side effects of the drug. In order to enter the target cell, the particle size of the polymeric nanocapsules must be below 20 nm or have targets that communicate with the proteins at the entrance of the cell.

[0003] Exosomes are a kind of naturally produced carriers that cells secrete to transport a wide range of diverse cargo, including DNA, RNA, proteins, and lipids, between various cells. Exosomes show promise in target therapy and therapeutic vectors due to their minimal immunogenicity. While exosomes have great potential in drug delivery systems, there may be challenges in their application in cancer treatment, such as low drug loading capacity, difficult and long-term extraction with low efficiency, allergies, low carrying capacity, isolation, and limited purification. Advanced technologies are required to achieve a novel exosome-based drug delivery system with advanced capabilities to overcome challenges. Therefore, according to what was mentioned, exosome and polymeric nanocapsules each have limitations that make targeted drug delivery associated with deficiencies. Thus, there isa need to develop a new nanocarrier structure based on the combination of exosome and polymeric nanocarrier to solve the limitations.SUMMARY

[0004] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more exemplary aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0005] One or more exemplary embodiments describe an exemplary targeted nanocarrier comprising an exemplary modified exosome. In an exemplary embodiment, an exemplary modified exosome may comprise an exemplary interior core comprising an exemplary aqueous medium of an exemplary modified exosome; and at least one exemplary bilayer membrane. In an exemplary embodiment, at least one exemplary bilayer membrane may comprise a plurality of exemplary phospholipid units; a plurality of exemplary amphiphilic copolymer units; and at least one exemplary assembled targeting ligand. One or more exemplary embodiments describe an exemplary pharmaceutical composition of cisplatin with a concentration between 0.01 mg / ml and 0.9 mg / ml. In an exemplary embodiment, an exemplary pharmaceutical composition may comprise a plurality of exemplary modified exosomes. In an exemplary embodiment, each of the plurality of exemplary modified exosomes may comprise an exemplary interior core comprising an exemplary aqueous medium, and at least one exemplary bilayer membrane. In an exemplary embodiment, an exemplary aqueous medium may comprise an exemplary solution of cisplatin molecules entrapped inside of an exemplary interior core.

[0006] In an exemplary embodiment, each exemplary phospholipid unit of the plurality of exemplary phospholipid units may comprise an exemplary first phospholipid subunit and an exemplary second phospholipid subunit. In an exemplary embodiment, each of exemplary first phospholipid subunit and exemplary second phospholipid subunit may comprise one exemplary polar end and two exemplary nonpolar ends. In an exemplary embodiment, each of exemplary nonpolar ends may comprise two exemplary hydrophobic tails. In an exemplary embodiment, each of one exemplary polar end may comprise an exemplary hydrophilic head. In an exemplary embodiment, exemplary polar ends may face towards an exemplary aqueous medium of an exemplary modified exosome. In an exemplary embodiment, exemplary nonpolar ends of an exemplary first phospholipid subunit of an exemplary phospholipid unit may face towards an exemplary corresponding second phospholipid subunit of an exemplary corresponding phospholipid unit. In an exemplary embodiment, exemplary nonpolar ends may face towards at least one exemplary bilayer membrane. In an exemplary embodiment, each of exemplary amphiphilic copolymer units may be assembled among exemplary phospholipid units with a predetermined weight ratio between 10: 100 and 100: 10 (exemplary phospholipid unit: exemplary copolymer unit). In an exemplary embodiment, each of exemplary amphiphilic copolymer units may comprise two exemplary hydrophilic block ends and one exemplary hydrophobic block unit. In an exemplary embodiment, each of exemplary hydrophobic block units may be assembled between two exemplary hydrophilic block ends. In an exemplary embodiment, each of two exemplary hydrophilic block ends may comprise at least two exemplary polymer chains. In an exemplary embodiment, exemplary hydrophilic ends may face towards an exemplary aqueous medium of an exemplary modified exosome. In an exemplary embodiment, exemplary hydrophobic block units may face towards inside an exemplary bilayer membrane. In an exemplary embodiment, exemplary hydrophobic block units of exemplary copolymer units and exemplary nonpolarunits of exemplary phospholipid units may create an exemplary hydrophobic space inside an exemplary bilayer membrane. In an exemplary embodiment, at least one exemplary assembled targeting ligand may be configured to identify a specific type of cell. In an exemplary embodiment, an exemplary modified exosome may have a diameter between 10 nm and 150 nm. In an exemplary embodiment, an exemplary amphiphilic copolymer unit may comprise (polyethylene glycol)2-block-polydimethylsiloxane-block-(polyethylene glycol)2((PEG)2-b-PDMS-b-(PEG)2). In an exemplary embodiment, each of two exemplary polymer chains may comprise polyethylene glycol with a molecular weight between 500 g / mol and 20000 g / mol. In an exemplary embodiment, an exemplary hydrophobic block unit may comprise polydimethylsiloxane.

[0007] This Summary may introduce a number of concepts in a simplified format; the concepts are further disclosed within the “Detailed Description” section. This Summary is not intended to configure essential / key features of the claimed subject matter, nor is intended to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following drawings in which an exemplary embodiment will now be illustrated by way of example. It is expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of one or more exemplary embodiments. One or more exemplary embodiments will now be described by way of example in association with the accompanying drawings in which:

[0009] FIG. 1 illustrates a schematic structure of an exemplary modified exosome, consistent with one or more exemplary embodiments of the present disclosure;

[0010] FIG. 2 illustrates a schematic structure of an exemplary modified exosome of an exemplary pharmaceutical composition comprising an exemplary hydrophilic drug, consistent with one or more exemplary embodiments of the present disclosure;

[0011] FIG. 3 illustrates a schematic structure of an exemplary modified exosome of an exemplary pharmaceutical composition comprising an exemplary hydrophobic drug, consistent with one or more exemplary embodiments of the present disclosure;

[0012] FIG. 4 illustrates an exemplary molecular structure of (polyethylene glycol)2-block-polydimethylsiloxane-block-(polyethylene glycol)2((PEG)2-b-PDMS-b-(PEG)2), consistent with one or more exemplary embodiments of the present disclosure;

[0013] FIG. 5 illustrates Atomic Force Microscopy (AFM) and High-Resolution Transmission Electron Microscopy (HRTEM) images of exemplary modified exosomes, consistent with one or more exemplary embodiments of the present disclosure;

[0014] FIG.6 illustrates a microfluidic process for producing an exemplary pharmaceutical composition comprising cisplatin by using a 3D micromixer system, consistent with one or more exemplary embodiments of the present disclosure;

[0015] FIG. 7 illustrates Encapsulation Efficiency (EE%) values obtained from different cisplatin starting concentration for two samples of exemplary modified exosomes, consistent with one or more exemplary embodiments of the present disclosure;

[0016] FIG. 8 illustrates cumulative drug cisplatin release profile, 7 days in the acidic environment with pHs = 5, 5.8, and 7.4 for sample of exemplary modified exosomes, consistent with one or more exemplary embodiments of the present disclosure;

[0017] FIG. 9 illustrates cytotoxic effect of an exemplary pharmaceutical composition comprising cisplatin measured by (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT), consistent with one or more exemplary embodiments of the present disclosure;

[0018] FIG. 10 illustrates Total Oxidant Status (TOS) level comparison between exemplary solution of cisplatin encapsulated inside of exemplary modified exosomes and other test groups, consistent with one or more exemplary embodiments of the present disclosure;

[0019] FIG. 11 illustrates Total Antioxidant Capacity (TAC) level comparison between exemplary solution of cisplatin encapsulated inside of exemplary modified exosomes and other test groups, consistent with one or more exemplary embodiments of the present disclosure;

[0020] FIG. 12 illustrates results of the gene expressions of IL-1, IL- 10, and IL- 12 in cells targeted with cisplatin; and

[0021] FIG. 13 illustrates x-CELLigence result for assessing efficacy of an exemplary solution of cisplatin entrapped inside of exemplary modified exosome, consistent with one or more exemplary embodiments of the present disclosure.DETAILED DESCRIPTION

[0022] In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings related to exemplary embodiments. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0023] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in one or more exemplary embodiments. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of one or more exemplary embodiments. However, it will be apparent to one skilled in the art that these specific details are not required to practice thedisclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to exemplary implementations will be plain to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of one or more exemplary embodiments. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0024] An exemplary embodiment is directed to an exemplary targeted nanocarrier for delivering drugs to targeted cells. In an exemplary embodiment, an exemplary targeted nanocarrier may comprise an exemplary modified exosome. Referring to figures, FIG. 1 illustrates a schematic structure of an exemplary modified exosome 100, consistent with one or more exemplary embodiments of the present disclosure. With further detail to FIG. 1, in an exemplary embodiment, an exemplary modified exosome 100 may comprise an exemplary interior core 102, and at least one exemplary bilayer membrane 104. In an exemplary embodiment, an exemplary interior core 102 may comprise an exemplary aqueous medium of an exemplary modified exosome 100. In an exemplary embodiment, an exemplary targeted nanocarrier comprising an exemplary modified exosome 100 may have a diameter between 10 nm and 150 nm.

[0025] An exemplary embodiment is directed to an exemplary pharmaceutical composition comprising an exemplary hydrophilic drug. In an exemplary embodiment, an exemplary pharmaceutical composition may comprise a plurality of exemplary modified exosomes, and an exemplary solution comprising a plurality of exemplary hydrophilic drug molecules. Referring to figures, FIG. 2 illustrates a schematic structure of an exemplary modified exosome 200 of an exemplary pharmaceutical composition comprising an exemplary hydrophilic drug, consistent with one or more exemplary embodiments of thepresent disclosure. With further detail to FIG. 2, in an exemplary embodiment, an exemplary modified exosome 200 may comprise an exemplary interior core 202, and at least one exemplary bilayer membrane 204. In an exemplary embodiment, an exemplary interior core 202 may comprise an exemplary aqueous medium of an exemplary modified exosome 200. In an exemplary embodiment, an exemplary solution comprising a plurality of exemplary hydrophilic drug molecules 206 may be entrapped inside an exemplary aqueous medium of an exemplary modified exosome 200. In an exemplary embodiment, an exemplary modified exosome 200 of an exemplary pharmaceutical composition comprising an exemplary hydrophilic drug may have a diameter between 10 nm and 150 nm.

[0026] An exemplary embodiment is directed to an exemplary pharmaceutical composition comprising an exemplary hydrophobic drug. In an exemplary embodiment, an exemplary pharmaceutical composition may comprise a plurality of exemplary modified exosomes, and a plurality of exemplary hydrophobic drug molecules. Referring to figures, FIG. 3 illustrates a schematic structure of an exemplary modified exosome 300 of an exemplary pharmaceutical composition comprising an exemplary hydrophobic drug, consistent with one or more exemplary embodiments of the present disclosure. With further detail to FIG. 3, in an exemplary embodiment, an exemplary modified exosome 300 may comprise an exemplary interior core 302, and at least one exemplary bilayer membrane 304. In an exemplary embodiment, an exemplary interior core 302 may comprise an exemplary aqueous medium of an exemplary modified exosome 300. In an exemplary embodiment, a plurality of exemplary hydrophobic drug molecules 306 may be entrapped inside an exemplary bilayer membrane 304 of an exemplary modified exosome 300. In an exemplary embodiment, an exemplary pharmaceutical composition comprising an exemplary hydrophobic drug 306 may comprise an exemplary solution of exemplary hydrophobic drug molecules 306 entrapped inside an exemplary bilayer membrane 304 of an exemplarymodified exosome 300. In an exemplary embodiment, an exemplary modified exosome 300 of an exemplary pharmaceutical composition comprising an exemplary hydrophobic drug may have a diameter between 10 nm and 150 nm.

[0027] An exemplary embodiment, is directed to an exemplary pharmaceutical composition comprising cisplatin. In an exemplary embodiment, an exemplary pharmaceutical composition may comprise a plurality of exemplary modified exosomes, and an exemplary solution comprising a plurality of cisplatin molecules. Referring to figures, FIG. 2 illustrates a schematic structure of an exemplary modified exosome 200 of an exemplary pharmaceutical composition comprising cisplatin, consistent with one or more exemplary embodiments of the present disclosure. With further detail to FIG. 2, in an exemplary embodiment, an exemplary modified exosome 200 may comprise an exemplary interior core 202, and at least one exemplary bilayer membrane 204. In an exemplary embodiment, an exemplary interior core 202 may comprise an exemplary aqueous medium of an exemplary modified exosome 200. In an exemplary embodiment, an exemplary aqueous medium of an exemplary modified exosome 200 may comprise an exemplary solution comprising a plurality of cisplatin molecules. In an exemplary embodiment, a plurality of cisplatin molecules 206 may be entrapped inside an exemplary aqueous medium of an exemplary modified exosome 200. In an exemplary embodiment, an exemplary pharmaceutical composition comprising cisplatin may comprise an exemplary solution of cisplatin molecules 206 entrapped inside an exemplary aqueous medium of an exemplary interior core 202. In an exemplary embodiment, an exemplary modified exosome 200 of an exemplary pharmaceutical composition comprising cisplatin may have a diameter between 10 nm and 150 nm.

[0028] In an exemplary embodiment, at least one exemplary bilayer membrane (104, 204, and 304) may comprise a plurality of exemplary phospholipid units (108, 208, and 308),a plurality of exemplary amphiphilic copolymer units (110, 210, and 310), and at least one exemplary assembled targeting ligand (112, 212, and 312). In an exemplary embodiment, each exemplary phospholipid unit (108, 208, and 308) of the plurality of exemplary phospholipid units (108, 208, and 308) may comprise an exemplary first phospholipid subunit (114, 214, and 314) and an exemplary second phospholipid subunit (116, 216, and 316). In an exemplary embodiment, each of an exemplary first phospholipid subunit (114, 214, and 314) and an exemplary second phospholipid subunit (116, 216, and 316) may comprise one exemplary polar end and two exemplary nonpolar ends. In an exemplary embodiment, each of exemplary nonpolar ends may comprise two exemplary hydrophobic tails. In an exemplary embodiment, each of two exemplary hydrophobic tails may be made of fatty acid chains. In an exemplary embodiment, one exemplary one polar end may comprise an exemplary hydrophilic head. In an exemplary embodiment, an exemplary hydrophilic head may comprise a phosphate group. In an exemplary embodiment, exemplary polar ends may be faced towards of an exemplary aqueous medium of an exemplary modified exosome (100, 200, and 300). In an exemplary embodiment, exemplary nonpolar ends of an exemplary first phospholipid subunit (114, 214, and 314) of an exemplary phospholipid unit (108, 208, and 308) may be faced towards of an exemplary corresponding second phospholipid subunit (116, 216, and 316) of an exemplary corresponding phospholipid unit (108, 208, and 308). In an exemplary embodiment, exemplary nonpolar ends may be faced towards of an exemplary bilayer membrane (104, 204, and 304).

[0029] In an exemplary embodiment, each of exemplary amphiphilic copolymer units (110, 210, and 310) may be assembled among phospholipid units (108, 208, and 308) with a predetermined weight ratio between 10:100 and 100:10 (exemplary phospholipid unit: exemplary copolymer unit). In an exemplary embodiment, each of exemplary amphiphilic copolymer unit (110, 210, and 310) may be configured in an exemplary H-shape structure.In an exemplary embodiment, each of exemplary amphiphilic copolymer units (110, 210, and 310) may comprise two exemplary hydrophilic block ends and an exemplary hydrophobic block unit. In an exemplary embodiment, each of exemplary hydrophobic block units may be assembled between exemplary hydrophilic block ends. In an exemplary embodiment, each of exemplary two hydrophilic block ends may comprise at least two exemplary polymer chains. In an exemplary embodiment, exemplary hydrophilic ends may be faced towards of an exemplary aqueous medium of an exemplary modified exosome (100, 200, and 300). In an exemplary embodiment, exemplary hydrophobic block units may be faced towards inside of an exemplary bilayer membrane (104, 204, and 304). In an exemplary embodiment, exemplary hydrophobic block units of exemplary copolymer units (110, 210, and 310) and exemplary nonpolar units of exemplary phospholipid units (108, 208, and 308) may create an exemplary hydrophobic space inside of an exemplary bilayer membrane (104, 204, and 304). In an exemplary embodiment, at least one exemplary assembled targeting ligand (112, 212, and 312) may be configured in an exemplary bilayer membrane (104, 204, and 304) to target a specific type of cells.

[0030] Copolymers containing both hydrophilic and hydrophobic chains are known as amphiphilic polymers. The ability to self-assemble in certain solvents is a special characteristic of this particular class of polymers. The polymer chains' hydrophilic-hydrophobic interactions, which result in micellar structures at the nano and micro scales, cause this behavior. Self-assembly gives rise to certain unusual qualities, such as structural polymorphism and thermos -gelling capabilities. Amphiphilic copolymers' favorable characteristics made them suitable for a range of uses, including those in the biomedical area such as drug delivery, gene therapy carriers, and cell encapsulation. They are the best options for drug delivery applications because of their amphiphilic character, which allowshydrophobic medicines to be delivered in an aqueous medium by being encapsulated in hydrogel micelles.

[0031] In an exemplary embodiment, an exemplary amphiphilic copolymer unit (110, 210, and 310) may comprise (polyethylene glycol)2-Z> / ock-polydimethylsiloxane-Z? / ock-(poly ethylene glycol (PEG)2-Z>-PDMS-Z?-(PEG)2. In an exemplary embodiment, each of two exemplary polymer chains may comprise polyethylene glycol with a molecular weight between 500 g / mol and 20000 g / mol. In an exemplary embodiment, an exemplary hydrophobic block unit may comprise poly dimethylsiloxane. Referring to figures, FIG. 4 illustrates an exemplary molecular structure of (PEG)2-Z>-PDMS-Z?-(PEG)2, consistent with one or more exemplary embodiments of the present disclosure. With further detail to FIG.4, (PEG)2- -PDMS-Z?-(PEG)2 may have a non-linear and H- shaped structure. With further detail to FIG.4, (PEG)2- -PDMS-Z?-(PEG)2 may have a conjugated structure of polyethylene glycol and poly dimethylsiloxane. In an exemplary embodiment, (PEG)2-Z>-PDMS-Z?-(PEG)2 may have an average molecular weight between 2000 g / mol and 20000 g / mol.

[0032] In an exemplary embodiment, each of exemplary phospholipid unit (108, 208, and 308) may be assembled between exemplary two amphiphilic copolymer units (110, 210, and 310). In an exemplary embodiment, each of amphiphilic copolymer unit (110, 210, and 310) may be assembled between two exemplary phospholipid units (108, 208, and 308). In a process of production of an exemplary modified exosome (100, 200, and 300), exemplary phospholipids units and exemplary amphiphilic copolymer units may be self-assembled to an exemplary shelf-gated polymeric vesicle, that an exemplary aqueous medium may be surrounded with bilayer membrane (104, 204, and 304).

[0033] “Nanocarrier” or “nanoencapsulation” or “nanodevice” or “nanoparticle” or “nanoscale drug delivery system” or “nanoscale encapsulation” or “nano capsule” or “nanosized capsule” or “nano-scale carrier” or “nano-scale encapsulated particle” may refer to tinytransport particles that can have their size, charge, and shape changed to deliver medicinal substances to particular regions. In addition to providing benefits including defense against drug deterioration, increased concentration in target tissues, and less harmful side effects, they serve as transport carriers, influencing the distribution and pharmacokinetics of pharmaceuticals. To improve their selectivity to particular target tissues, nanocarriers can also be coupled to particular ligands.

[0034] Exosome is naturally present in cells with a lipid vesicle structure, on the surface of which there are proteins that can easily enter the cell with a maximum size of 150 nm. Exosomes cause the growth, increase angiogenesis and the transfer of cancer cells to other parts of the body, for this reason, cancer cells have a great tendency to absorb exosomes, and as a result, the use of proteins in exosomes as targeting causes increased absorption and easy entry of nanocarriers into cancer cells. This new nanocarrier may have a pH-sensitive delivery feature and are an excellent candidate for intracellular drug delivery systems as a result of the autophagy phenomenon. The H- shaped structure of exemplary amphiphilic copolymer may be similar to the bilayer lipid structure of cells, and exemplary block units in exemplary amphiphilic copolymer may have a high flexibility, transparency, and biocompatibility. These nanocarriers with a uniform and controlled size with a hydrodynamic diameter (HD) of less than 20 nm for one-way facilitated passage through blood barriers are very suitable for the effective treatment of all types of cancer, especially brain cancer, which have a better effect in vivo and in vitro than other nanocarriers.

[0035] With further detail to FIG.5, FIG.5 illustrates Atomic Force Microscopy (AFM) 502 and High-Resolution Transmission Electron Microscopy (HRTEM) 504 images of exemplary modified exosomes, consistent with one or more exemplary embodiments of the present disclosure. The mean diameter of an exemplary modified exosome was 20 + 5 nm for this specimen. Core of an exemplary modified exosome was light and the shell dark areas.EXAMPLES

[0036] Hereinafter, one or more exemplary embodiments will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples may be for illustrative purposes only and are not to be interpreted to limit the scope of one or more exemplary embodiments. It must be understood that in the following examples (Example 1-6), the abbreviation of PEG is used instead of polyethylene glycol, PDMS is used instead of poly (poly dimethylsiloxane), THF is used instead of Tetrahydrofuran, DMF is used instead of Dimethylformamide, and UV is used instead of Ultraviolet rpm. Moreover, it must be understood that in the following examples (Example 1-6), group A is used instead of group “control”, group B is used instead of group “Nanoparticles of copolymer (2mg / ml)”, group C is used instead of group “Targeted nanocarrier comprising modified exosome (lmg / ml)”, group D is used instead of group “Free form of cisplatin (100pg / ml)”, group E is used instead of group “Nanoparticles of copolymer bound cisplatin (100pg / ml)”, group F is used instead of group “Nanoparticles of copolymer bound cisplatin (50pg / ml)”, group G is used instead of group “Nanoparticles of copolymer bound cisplatin (25pg / ml)”, group H is used instead of group “Nanoparticles of copolymer bound cisplatin (12.5pg / ml)”, group I is used instead of group “A solution of cisplatin (lOOpg / mlv) entrapped inside of modified exosome (100pg / ml)”, group J is used instead of group “A solution of cisplatin (50pg / mlv) entrapped inside of modified exosome (50pg / ml)”, group K is used instead of group “A solution of cisplatin (25pg / mlv) entrapped inside of modified exosome (25pg / ml)”, and group L is used instead of group “A solution of cisplatin (12.5pg / mlv) entrapped inside of modified exosome (12.5pg / ml)”.Example 1: Producing an exemplary pharmaceutical composition of cisplatin

[0037] The exosomes were extracted from T98G cells and were dissolved in a mixture of chloroform-ethanol with a volume ratio of 50:50 (chloroform: ethanol) to form a solutionof exosomes with a concentration of 0.5 mg / ml. Simultaneously, synthesized PEG1000-PDMS550-PEG1000copolymer was dissolved in a mixture of THF-DMF with a volume ratio of 80:20 (THF: DMF) to form a solution of PEG1000-PDMS550-PEG1000copolymer with a concentration of 0.5 mg / ml and the two formed solutions were mixing together. After that, a powder of cisplatin with a final concentration between 0.01 mg / ml and 0.9 mg / ml was added to the mixed solution and diluted by DMF. FIG.6 illustrates a microfluidic process 600 for producing an exemplary pharmaceutical composition comprising cisplatin by using a 3D micromixer system, consistent with one or more exemplary embodiments of the present disclosure. The diluted solution was injected to the 3D micromixer system as shown in FIG.6. The 3D micromixer's output solution is injected into the microfluidic device at a flow rate of 5 pl / min, and as an anti-solvent, water is injected through inlets (a) at a flow rate of 100 pl / min. Consequently, the confluence of two fluids at region (d) caused the self-assembly phenomena to begin, which in turn caused the production of exemplary modified exosomes comprising cisplatin through on-chip self-assembly. To obtain the same small amount of the solution, argon gas was introduced into the channel through the region of (e). Eventually, processing channel (f), which had a length of 740 mm, regulated the size of the particles. Micelles swiftly cycled through the processing channel in the small plugs, and in a gas-liquid microfluidic reactor, the competition between shear-induced particle broke down and shear-induced coalescence might adjust the ultimate particle size.Example 2: Encapsulation Efficiency and release properties of an exemplary pharmaceutical composition of cisplatin

[0038] Cisplatin served as the model medication. Therefore, a 10 pg / mL cisplatin solution in phosphate buffer pH 6.8 was produced for the Calibration curve, and various volumes, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mL were extracted from this solution. Then, in order to obtain a light green color solution, 1 mL of 12-oxo Phytodienoic acid (OPDA) solution(1.4 mg / mL) and 2 mL of phosphate buffer pH 6.8 were added to each of these extracted solutions and heated at 100 °C for 10 minutes. Following the cooling of the green color solutions to room temperature, 10 mL of the solutions were prepared using DMF, and a UV-Visible spectrophotometer was used to measure the solutions' UV spectra at 706 nm. In order to evaluate the cisplatin loading capacity and release characteristics of an exemplary modified exosome, a microfluidic device was constructed to produce exemplary modified exosomes containing a 10 pg / mL solution of cisplatin in phosphate buffer pH 6.8. This procedure is detailed in Example 1. Exemplary modified exosomes were first put into a dialysis bag and immersed in phosphate buffer (pH 6.8) in order to evaluate loading. Three times, at ten-minute intervals, the buffer was switched out for new buffer. The dialysis bag solution was then subjected to three 10-minute ultrasonic baths to break up exosomes and release loaded cisplatin into the solution. To obtain a light green color, the final solution was heated to 100 °C for 10 minutes. After cooling, 10 mL of the solution was prepared using DMF, and the UV spectrum was recorded at 706 nm. The cisplatin-containing modified exosomes were put into a dialysis bag, immersed in three different buffer solutions (pHs 5, 5.8, and 7.4), and then placed in an incubator at 37 °C with gradual agitation (100 rpm) for the purpose of studying the release of cisplatin from exemplary modified exosomes. Up to a week, aliquots were obtained from each solution at various intervals. All sample's UV spectra were measured at 706 nm. FIG. 7 illustrates Encapsulation efficiency (EE%) values 700 obtained from different cisplatin starting concentration for two samples of exemplary modified exosomes, consistent with one or more exemplary embodiments of the present disclosure.FIG. 8 illustrates cumulative drug cisplatin release profile 800, 7 days in the acidic environment with pHs = 5, 5.8, and 7.4 for sample of exemplary modified exosomes, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 7, exemplary modified exosome had high EE% compare to exemplary nanoparticle ofcopolymer. As shown in FIG. 8, cumulative release of exemplary modified exosome was higher than exemplary nanoparticle of copolymer.Example 3: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay

[0039] In short, 10 pL of MTT reagent was applied to each well and incubated for 4 hours at 37 °C with 5% CO2. After removing the medium, each well received 100 pL of dimethyl sulfoxide. Using a MultiskanTM GO Microplate Spectrophotometer reader (Thermo Scientific, Canada, USA), the optical density was measured at 570 nm. The viability % ratio formula was used to calculate the cell viability (%). FIG.9 illustrates cytotoxic effect 900 of an exemplary pharmaceutical composition of cisplatin measured by MTT, consistent with one or more exemplary embodiments of the present disclosure. After 24 hours of cell exposure, all dosages using this method produced a similar or higher level of cell death. Equivalent cell death, or in the event that pure Cisplatin was administered, was noted at the lowest dosage of exemplary modified exosome comprising cisplatin (12.5 pg / mL). The cytotoxic impact on the cancer line grew more evident when the dose of exosome and polymer nanoparticles was increased, which went against the predicted cytotoxic effects of Cisplatin encapsulated inside of exemplary exosome. The findings showed that, in comparison to the control, exemplary modified exosome comprising 100 pg / mL of Cisplatin and exemplary polymer nanoparticles carrying 100 pg / mL of Cisplatin reduced cell viability by 60% and 35%, respectively. The findings demonstrated the effectiveness of both exemplary modified exosome and exemplary polymer nanoparticles containing cisplatin at all dosages; however, exemplary modified exosome of cisplatin outperformed exemplary polymer nanoparticles at all levels between 12.5 and 100 pg / ml.Example 4: Total oxidant status (TOS) and Total Antioxidant Capacity (TAC)

[0040] The protocol for the commercial manufacturing kit was followed while performing the TOS assay. In summary, wells were filled with 500 pl of Reactive 1 solution, and the absorbance was measured at 530 nm initially. Next, the same well was filled with 25 pl of Reactive 2 solution, and the absorbance at 530 nm was measured again. TOS levels were determined as mmol Trolox equiv / mmol-1. The evaluation was completed with the formula (1). 5 ST2 (5 standard 2 = ST2 second reading - ST2 first reading), 5 Sample (5 Sample= Sample second reading- Sample first reading)

[0041] Formula

[0042] FIG. 10 illustrates TOS level comparison 1000 between exemplary solution of cisplatin encapsulated inside exemplary modified exosomes and other test groups, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 10, TOS 1000 for all groups was increased compared with control. Moreover, by increasing the amount of modified exosome, TOS was increased. The maximum value of TOS (5 H2O2mmol / L) was measured for the group I which was significantly higher than that of free form of Cisplatin in dose of 100 pg / mL (1.9 H2O2mmol / L) administered for neuroblastoma cells.

[0043] As directed by the commercial manufacturing kit method, the TAC test was performed. In summary, wells were filled with 500 pl of Reactive 1 solution, and the first absorbance was measured at 660 nm. After adding 75 pl of Reactive 2 to the identical wells, the absorbance value at 660 nm was measured for the second time. The TAC concentrations were given in mmol equiv / mmol-1. The evaluation was completed with the formula (2). (5 STI (5 standard 1 = STI second reading - STI first reading), 5 ST2 (5 standard 2 = ST2 second reading - ST2 first reading), 5 Sample (5 Sample = Sample second reading- Sample first reading).

[0044] Formula

[0045] FIG. 11 illustrates TAC level comparison 1100 between exemplary solution of cisplatin encapsulated inside exemplary modified exosomes and other test groups, consistent with one or more exemplary embodiments of the present disclosure. TAC, or serum total antioxidant capacity, is one of the many cancer biomarkers being studied under various neoplastic conditions. As shown in FIG. 11, When compared to the control, serum TAC was considerably lower in the group I. In comparison to the control group level of 5.2 Trolox mmol / L, the TAC levels for the solution of cisplatin entrapped inside modified exosome at two doses of 50 and 100 pg / mL were dropped to 2.8 and 2.1 (Trolox mmol / L) and 3.5 and 3.1 (Trolox mmol / L), respectively.Example 5: Gene Expression in Neuroblastoma

[0046] FIG. 12 illustrates results of the gene expressions of IL-1 (1202), IL- 10 (1204), and IL- 12 (1206) in cells targeted with cisplatin. As shown in FIG. 12, IL-1 (1202) and IL-12 (1204) expressions was significantly lower in all four group of exemplary solutions of cisplatin entrapped inside of exemplary modified exosome (I, J, K, and L) compared with control. Moreover, the IL-1 and IL- 12 expressions in the group of nanoparticles of copolymer bound cisplatin (E, F, G, and, H) was decreased by increasing the concentration of cisplatin. In group I, J, K, and L, the values decreased by increasing the concentration of cisplatin and exosome such that for the group I with dose of 100 pg / mL it decreased down to 4.33, while exemplary targeted nanocarrier comprising exemplary modified exosome, the corresponding value was 22.34 that is more than the control.Example 6: x-CELLigence result for assessing efficacy of an exemplary solution of cisplatin entrapped inside of exemplary modified exosome

[0047] Using x-Celligence technology, the profiling activity of the neuroblastoma cancer cells was examined in real time for the different states of cisplatin in order to assess the efficacy of the drug delivered into the cells by exemplary modified exosomes. FIG. 13illustrates x-CELLigence result 1300 for assessing efficacy of an exemplary solution of cisplatin entrapped inside of exemplary modified exosome, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 13, Cisplatin's ability to lower cell index is improved when it was entrapped in a polymer shell and its concentration was increased. Additionally, a considerable decrease in cell index was observed for an exemplary solution of cisplatin entrapped inside of exemplary modified exosome, as evidenced by the curves for these samples, which peaked in the 20-30 h time window before significantly declining in cell index values. Following a 96-hour period, the cell viability of all four group of exemplary solutions of cisplatin entrapped inside of exemplary modified exosome (I, J, K, and L) was nearly completely reduced, as seen by the cell index values of less than 1 and 0.46 for and exemplary solutions of cisplatin entrapped inside of exemplary modified exosome with a concentration of 100 pg / mL of cisplatin.

[0048] While the foregoing has described what are considered to be the best mode and / or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0049] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0050] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaningof the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0051] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0052] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0053] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0054] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study, except where specific meanings have otherwise beenset forth herein. Relational terms such as “first” and “second” and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0055] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it may be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0056] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and / or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

What is claimed is:

1. A targeted nanocarrier comprising a modified exosome, the modified exosome comprising: an interior core comprising an aqueous medium of the modified exosome; andat least one bilayer membrane comprising:a plurality of phospholipid units, each phospholipid unit of the plurality of phospholipid units comprising a first phospholipid subunit and a second phospholipid subunit, each of the first phospholipid subunit and the second subunit comprising one polar end and two nonpolar ends, each of the nonpolar ends comprising two hydrophobic tails, each of one polar end comprising a hydrophilic head, the polar ends facing towards the aqueous medium of the modified exosome, the nonpolar ends of the first phospholipid subunit of the phospholipid unit facing towards the corresponding second phospholipid subunit of the corresponding phospholipid unit, the nonpolar ends facing towards at least one bilayer membrane;a plurality of amphiphilic copolymer units, each of the amphiphilic copolymer units assembling among the phospholipid units with a predetermined weight ratio between 10: 100 and 100:10 (the phospholipid unit: the copolymer unit), each of the amphiphilic copolymer units comprising two hydrophilic block ends and one hydrophobic block unit, each of the hydrophobic block units assembling between two hydrophilic block ends, each of two hydrophilic block ends comprising at least two polymer chains, the hydrophilic ends facing towards the aqueous medium of the modified exosome, the hydrophobic block units facing towards the inside of the bilayer membrane, the hydrophobic block units of the copolymer units and the nonpolar units of the phospholipid units creating a hydrophobic space inside the bilayer membrane; andat least one assembled targeting ligand, wherein at least one assembled targeting ligand is configured to identify a specific type of cell.

2. The targeted nanocarrier of claim 1, wherein the targeted nanocarrier has a diameter between 10 nm and 150 nm.

3. The targeted nanocarrier of claim 1, wherein the modified exosome further comprises a solution comprising a plurality of hydrophobic drug molecules entrapped between two layers of the bilayer membrane.

4. The targeted nanocarrier of claim 1, wherein the modified exosome further comprises a solution comprising a plurality of hydrophilic drug molecules entrapped inside the interior core.

5. The targeted nanocarrier of claim 1, wherein the amphiphilic copolymer unit comprises (polyethylene glycol)2--polydimethylsiloxane--(polyethylene glycol)2((PEG)2--PDMS--(PEG)2).

6. The targeted nanocarrier of claim 1, wherein each of two polymer chains comprises polyethylene glycol with a molecular weight between 500 g / mol and 20000 g / mol.

7. The targeted nanocarrier of claim 1, wherein the hydrophobic block unit comprises Polydimethylsiloxane.

8. A pharmaceutical composition of cisplatin with a concentration between 0.9 mg / ml and 0.01 mg / m, the pharmaceutical composition comprising a plurality of a modified exosomes, each of the plurality of the modified exosomes comprising:an interior core comprising an aqueous medium, the aqueous medium comprising a solution of cisplatin molecules entrapped inside of the interior core; andat least one bilayer membrane comprising:a plurality of phospholipid units, each phospholipid unit of a plurality of phospholipid units comprising a first phospholipid subunit and a second phospholipid subunit, each of the first phospholipid subunit and the second subunit comprising one polar end and two nonpolar ends, each of the nonpolar ends comprising two hydrophobic tails, each of one polar end comprising a hydrophilic head, the polar ends facing towards the aqueous medium of themodified exosome, the nonpolar ends of the first phospholipid subunit of the phospholipid unit facing towards the corresponding second phospholipid subunit of the corresponding phospholipid unit, the nonpolar ends facing towards at least one bilayer membrane;a plurality of amphiphilic copolymer units, each of the amphiphilic copolymer units assembling among the phospholipid units with a predetermined weight ratio between 10: 100 and 100:10 (phospholipid unit: copolymer unit), each of the amphiphilic copolymer units comprising two hydrophilic block ends and one hydrophobic block unit, each of the hydrophobic block units assembling between two hydrophilic block ends, each of two hydrophilic block ends comprising at least two polymer chains, the hydrophilic ends facing towards the aqueous medium of the modified exosome, the hydrophobic block units facing towards the inside of the bilayer membrane, the hydrophobic block units of the copolymer units and the nonpolar units of the phospholipid units creating a hydrophobic space inside the bilayer membrane; andat least one assembled targeting ligand, wherein at least one assembled targeting ligand is configured to identify a specific type of cell.

9. The pharmaceutical composition of claim 8, wherein the solution of cisplatin molecules is surrounded by the bilayer membrane.

10. The pharmaceutical composition of claim 8, wherein the amphiphilic copolymer unit comprises (polyethylene glycol)2--polydimethylsiloxane--(polyethylene glycol)2(PEG)2--PDMS--(PEG)2.

11. The pharmaceutical composition of claim 8, wherein each of two polymer chains comprises polyethylene glycol with a molecular weight between 500 g / mol and 20000 g / mol.

12. The pharmaceutical composition of claim 8, wherein the hydrophobic block unit comprises polydimethylsiloxane.

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