A surface-functionalized, hepatocyte-specific exosomes, and methods of preparations thereof

Abstract

The present invention relates to surface-functionalized extracellular vehicles. Specifically, the present invention relates to exosomes surface-functionalized with lactosylated polyetheneimine (PEI-LA), such surface-functionalized exosomes as drug delivery vehicles, 5 compositions comprising a therapeutic agent encapsulated within such exosomes, methods of producing such exosomes and compositions thereof, as well as methods of delivering such exosomes and compositions to liver.

Description

A SURFACE-FUNCTIONALIZED, HEPATOCYTE-SPECIFIC EXOSOMES, AND METHODS OF PREPARATIONS THEREOFFIELD OF THE INVENTION

[0001] The present invention relates to surface-functionalized extracellular vehicles. Specifically, the present invention relates to surface-functionalized exosomes with ligandspecific polycations, such surface-functionalized exosomes as drug delivery vehicles, compositions comprising a therapeutic agent encapsulated within such exosomes, methods of producing such exosomes and compositions thereof, as well as methods of delivering such exosomes and compositions to a specific tissue or an organ.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Currently, prevalence of sedentary lifestyle, exposure to drugs and food toxins are becoming the major contributors to liver-related diseases worldwide. Despite the prevalence, very few effective pharmacological options are available for emergency situations to cure or prevent the worsening of liver functions.

[0004] Many researchers have inferred the potential of stem cells-derived exosomes in recovering the functional aspects of failing liver. While many of these therapeutic agents suffer poor efficacy, pharmacokinetics or off-target side effects due to systemic administration. One means of addressing these limitations is through targeted exosomal delivery utilizing a minimally altering surface modification. This will allow these modified exosomes to target the liver passively or actively (via ASGPR recognition), thereby reducing systemic circulation and increasing the “effective dose” in the liver. While few studies have addressed these gaps however, the use of chemical modification within the surface of these bilayered lipidic nanoparticles makes it a complicated solution. Our exosome modification approach is simple, ready to use and minimally alters the surface composition. Thus, giving it an edge over other formulations as our system can be easily prepared just before their use. Extensive in-vitro and in-vivo studies have provided the proof-of concept for the efficacy of this novel approach towards targeting of liver cells by modified exosomes and can be easily translated to human applications.

[0005] Every year liver-related diseases accounts for 2 million deaths, such that 1 out of every 25 deaths worldwide is caused by liver complications. At present, it has become the 11th leading cause for death worldwide. The increasing prevalence of liver diseases is driven by factors such as the aging global population, changing lifestyle patterns, and increased consumption of packaged foods, which have collectively spurred investment in research and development for liver therapeutics. The global liver disease treatment market was valued at US $21 billion in 2020 and is projected to grow at a compound annual growth rate (CAGR) of around 7% from 2021 to 2030. Among the various liver conditions, drug-induced liver injury (DILI) is a significant cause of acute liver failure and acute hepatitis, often resulting from long-term treatments for conditions like tuberculosis, cancer, epilepsy, and immune suppression. Due to this, the hepatoprotective drug market is expected to develop revenue and exponential market growth at a remarkable CAGR during the forecast period of 2023- 2030.

[0006] However, despite the potential benefits, the preventive use of hepatoprotective drugs is not yet widely recommended due to insufficient evidence supporting their efficacy in preventing DILI. The global burden of liver diseases is further compounded by the high prevalence of conditions such as non-alcoholic fatty liver disease (NALLD), cirrhosis, and hepatocellular carcinoma, with viral hepatitis and alcohol being major contributors. Notably, alcohol-associated liver disease (ALD) remains a leading cause of liver-related mortality, particularly among the productive-age population, exacerbated by patterns of heavy drinking and insufficient public health policies. Effective strategies for liver disease control and prevention must consider these diverse etiologies and risk factors, emphasizing early detection, lifestyle modifications, and targeted therapeutic interventions.

[0007] The liver's vital functions, such as drug metabolism, detoxification, and bioactive molecule synthesis, make it susceptible to chronic diseases, which often lack effective treatment options. Mesenchymal stem cell-derived exosomes are emerging as promising delivery vehicles due to their low immunogenicity and customizable surfaces. However, systemic injections face challenges, including poor targeting and rapid clearance from the body.

[0008] CN104264479B discloses a preparation method that may be used for capturing the lactobionic acid functionalized nano-fiber for capturing cancerous cell. The coupling reaction is between Lactobionic acid and polyethylene glycol and separately polyethyleneimine is blended with polyvinyl alcohol to form nano-fibers and then the two formulations are used.

[0009] CN100549044C reports the crosslinking of lactobionic acid with polyethyleneimine to form galactosyl polyethyleneimine that will be used in DNA transfection.

[0010] KR20120097865A describes a manufacturing method of the copolymer comprises a step of synthesizing polyethylene glycol by reacting galactose-containing glycoprotein, and a bifunctional polyethylene glycol; a step of manufacturing a chitosan- graft-spermine copolymer by reacting chitosan and spermine; and a step of reacting the galactosylated polyethylene glycol and the chitosan-graft-spermine copolymer.

[0011] However, the methods given in the prior arts are tedious and have their own limitations. Also, such methods have not been used for creating exosome-based hepatocytespecific polymer -exosome nanoconjugates. To overcome these limitations, the inventors have developed a novel delivery method for creating hepatocyte-specific polymer-exosome nanoconjugates synthesized through reductive amination in an aqueous medium.

[0012] The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.OBJECTS OF THE INVENTION

[0013] Objects of the present invention is to provide a drug-delivery system for targeted delivery in challenging physiological environments.

[0014] An object of the present invention is to provide hepatocyte-specific surface- functionalized exosomes.

[0015] An object of the present invention is to provide a method of preparing hepatocyte-specific surface-functionalized exosomes.

[0016] Another object of the present invention is to provide a hepatoprotective composition comprising the hepatocyte-specific surface-functionalized exosomes.

[0017] Yet another object of the present invention is to provide a method of treating liver diseases using the hepatoprotective composition comprising the hepatocyte-specific surface- functionalized exosomes.SUMMARY OF THE INVENTION

[0018] This summary is provided to introduce a selection of concepts in a simplified form that is further described below in the detailed description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0019] Aspects of the present invention relate to surface-functionalized extracellular vehicles. Specifically, the present invention relates to surface-functionalized exosomes with ligand-specific polycations, such surface -functionalized exosomes as drug delivery vehicles, compositions comprising a therapeutic agent encapsulated within such exosomes, methods of producing such exosomes and compositions thereof, as well as methods of delivering such exosomes and compositions to a specific tissue or an organ.

[0020] In another aspect, the present invention provides surface-functionalized exosomes for targeted delivery. In some embodiments, the surface-functionalized exosome is a hepatocyte-targeted exosome presenting a targeting moiety on its surface, wherein the targeting moiety is the PEI-LA. The surface modification forms a stable, reproducible vesicle complex in aqueous environments, resistant to aggregation and proteolytic degradation. This approach enhances bioavailability, targeting precision, and therapeutic stability of the vesicles when administered intravenously.

[0021] In another aspect, the present invention provides a method of preparing the hepatocyte-targeted exosome comprising the steps of: a) providing stem cells-derived exosomes; and b) incubating the stem cells-derived exosomes with the PEI-LA for up to 5 minutes in presence of NaCl solution or basal culture media to obtain the hepatocyte-targeted exosome; and c) concentrating the hepatocyte-targeted exosome by centrifugation using ultrafilters

[0022] In another aspect, the present invention provides a one-pot method of synthesizing lactosylated polyetheneimine (PEI-LA) by reductive amination comprising: a) preparing lactobionic acid (LA) solution in water at pH 5; b) adding l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) to the LA solution, and immediately followed by N-hydroxy succinimide (NHS) to the LA solution to obtain a reaction mixture; c) incubating the reaction mixture for upto 30 min at room temperature; d) adding branched polyethyleneimine (PEI) dropwise to the incubated reaction mixture from step c), followed by incubating the same for up to 48 hrs to obtain PEI-LA in solution; and e) dialyzing the solution comprising the PEI-LA for upto 48 hrs with 3kDa membrane in distilled water to obtain a purified PEI-LA.

[0023] In another aspect, the present invention provides a lactosylated polyetheneimine (PEI-LA) prepared by the method as disclosed herein.

[0024] In yet another aspect, the present invention provides a drug-delivery system for targeted delivery to hepatocytes comprising the hepatocyte-targeted exosome as disclosed herein and a hepatoprotective drug.

[0025] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.BRIEF DESCRIPTION OF THE DRAWINGS:

[0026] FIG. 1 represents the Synthesis and characterisation of ASGPR-specific polymer. (A) Reaction scheme for the synthesis of lactosylated polyethyleneimine (PEI-LA), (B) NMR and (C) FTIR analysis of the synthesized PEI-LA conjugates, (D) Linear plot showing increasing concentration of galactose sugar in the PEI-LA conjugates, (E) Cell viability assay on HepG2 after 4h incubation of PEI-LA conjugates.

[0027] FIG. 2 represents the Size and morphological characterisation of BMSC-derived exosomes and their modification. (A) Schematic illustration of the procedure to fabricate exosome-polymer nanocomplexes. Size and morphological comparative analysis of exosomes and modified exosomes using (B) DLS, (C) AFM, (D) NTA, (E) SEM, TEM and Immunogold TEM.

[0028] FIG. 3 represents the in vitro analysis of modified exosome uptake efficacy. Calcein AM-labelled (green) exosome and modified exosome uptake kinetics through (A) confocal imaging and (B) flow cytometry at different time intervals. (C) Percent fluorescence intensity of Calcein-AM labelled exosomes with respect to parent population. (D) In vivo biodistribution of Pkh-26 (red) exosome and modified exosome in the healthy rat liver, imaged using confocal microscopy.

[0029] FIG. 4 represents the hepatoprotective effect of modified exosomes during acetaminophen-induced acute liver injury rat model. In vivo analysis for acetaminopheninduced acute liver failure model, showing (A) digital images of the liver, (B) H and E staining, and (C) expression of caspase 3 in the liver sections.

[0030] FIG. 5 represents the efficacy of modified exosomes for liver regeneration after acetaminophen-induced acute-on-chronic liver failure. In vivo analysis shows the (A) digital images of the liver, (B) H and E staining, and (C) Picrosirius red (PSR) staining of the liver sections, where cell apoptosis, ECM deposition and eventual cell proliferation can be seen.

[0031] FIG. 6 represents the effect of exosomes on liver and bone metabolism in a CCh induced hepatic dystrophy model. In vivo analysis of the chronic liver harvested from the different groups: (A) Representative digital images with different degrees of injuries and resolution, (B) HE and PSR images revealing the fibrotic briges, cell necrosis, ballooning and collagen deposition within different groups, (C) Bone volume / Tissue volume ratios and (D) Peak fracture force of the femur revealing the bone mineralisation.DETAILED DESCRIPTION OF THE INVENTION

[0032] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

[0033] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0034] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0035] In some embodiments, numbers have been used for quantifying weight percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention areapproximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0036] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0037] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0038] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0039] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0040] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0041] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

[0042] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of theprinciples and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0043] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0044] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0045] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements a, b, and c, and a second embodiment comprises elements b and d, then the inventive subject matter is also considered to include other remaining combinations of a, b, c, or d, even if not explicitly disclosed.

[0046] While a particular form of the invention has been illustrated and described, it will be apparent that various modifications can be made without departing from the spirit and scope of the invention.

[0047] As described herein, the terms “Extracellular vesicles” and “exosome” are used interchangeably herein with the terms “exosome,” “exosome-like particle,” “exosome-like vesicle,” and grammatical variations of each of the foregoing.

[0048] As described herein, the term ‘effective amount’ refers to the amount of the formulation required to bring about a change or improvement in a subject without side effects or overdosing.

[0049] The term, "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class 'mammalia' , including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.

[0050] Embodiments of the present invention relate to surface-functionalized extracellular vehicles. Specifically, the present invention relates to surface-functionalized exosomes with ligand-specific polycations, such surface-functionalized exosomes as drug delivery vehicles, compositions comprising a therapeutic agent encapsulated within suchexosomes, methods of producing such exosomes and compositions thereof, as well as methods of delivering such exosomes and compositions to a specific tissue or an organ.

[0051] In an embodiment, the present invention provides exosomes whose surfaces are with ligand-specific polycations targeting asialoglycoprotein receptors of the hepatocytes.

[0052] In some embodiments of the present invention, the surface of the ligand-specific poly cation is lactosylated polyetheneimine (PEI-LA). In some embodiments, the combination of lactosylation with PEI modification on exosomes enhances targetability to liver cells or hepatocytes. The use of lactobionic acid conjugation specifically addresses the limitations of conventional exosome targeting methods by increasing tissue-specific internalization without altering the native properties of the exosomes.

[0053] In an embodiment, the present invention provides a one-step procedure to first functionalize polyethyleneimine with Lactobionic acid at room temperature in water.

[0054] In a preferred embodiment, the present invention provides a one-pot method of synthesizing lactosylated polyetheneimine (PEI-LA) by reductive amination comprising: a) preparing lactobionic acid (LA) solution in water at pH 5; b) adding l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) to the LA solution, and immediately followed by N-hydroxy succinimide (NHS) to the LA solution to obtain a reaction mixture; c) incubating the reaction mixture for upto 30 min at room temperature; d) adding branched polyethyleneimine (PEI) dropwise to the incubated reaction mixture from step c), followed by incubating the same for up to 48 hrs to obtain PEI-LA in solution; and e) dialyzing the solution comprising the PEI-LA for upto 48 hrs with 3kDa membrane in distilled water to obtain a purified PEI-LA.

[0055] In some embodiments of the present invention, the PEI has a size of 2000 - 25000 kDa

[0056] In some embodiments of the present invention, the PEI, LA, EDC, and NHS are used in a ratio of is 1 to 2:50 to 100:50 to 100:50 to 100, respectively. In a preferred embodiment, the ratio of PEI, LA, EDC, and NHS is 1:50: 100: 100, respectively.

[0057] In an embodiment, the present invention provides a lactosylated polyetheneimine (PEI-LA) prepared by the method as disclosed herein.

[0058] In another embodiment, the present invention provides surface-functionalized exosomes for targeted delivery. In some embodiments, the surface-functionalized exosome is a hepatocyte-targeted exosome presenting a targeting moiety on its surface, wherein thetargeting moiety is the PEI-LA. The surface modification forms a stable, reproducible vesicle complex in aqueous environments, resistant to aggregation and proteolytic degradation. This approach enhances bioavailability, targeting precision, and therapeutic stability of the vesicles when administered intravenously.

[0059] In some embodiments of the present invention, the PEI-LA is non-covalently conjugated to surface of the exosome. In a preferred embodiment, the non-covalent conjugation is by electrostatic interaction. Unlike traditional covalent modification methods that may alter exosomal structure or function, this non-covalent approach maintains the exosome's structural integrity and biological activity with minimal changes to its bilipid membrane, which is crucial for therapeutic efficacy.

[0060] In some embodiments of the present invention, the hepatocyte-targeted exosome as disclosed herein targets asialoglycoprotein receptors (ASGPR) on hepatocytes. The use of PEI-LA enables specific targeting to hepatocytes via the ASGPR, a strategy not previously reported with such a rapid and efficient formulation. This specificity enhances the bioavailability and therapeutic efficacy of the exosomes.

[0061] In some embodiments of the present invention, the hepatocyte -targeted exosomes are nanoparticles having average sizes ranging from at least about 1 nm, 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2500 nm, 3000 nm, 4000 nm, 5000 nm, 6000 nm, 7000 nm, 8000 nm, or at least 9000 nm. In some embodiments, the nanoparticles may have a diameter of less than 10,000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4500 nm, 4000 nm, 3500 nm, 3000 nm,2500 nm, 2000 nm, 1900 nm, 1800 nm, 1700 nm, 1600 nm, 1500 nm, 1400 nm, 1300 nm,1200 nm, 1100 nm, 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 250 nm, or less than 100 nm. The diameter of nanoparticles can range from any of the minimum values described above to any of the maximum values described above, for example from 1 nm to 10,000 nm, 50 nm to 5,000 nm, 10 nm to 250 nm, 20 nm to 200 nm, or 20 nm to 100 nm. Preferably 200 nm to 400 nm.

[0062] In an embodiment of the present invention, the hepatocyte-targeted exosomes exhibit a zeta potential of -10 to -15 mV. Preferably, -10±2.3 mV.

[0063] In an embodiment of the present invention, the hepatocyte-targeted exosomes exhibit a hepatocyte internalization of upto 50-80%. For example, 50%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%. 76%, 77%, 78%, 79%, or 80%.

[0064] In an embodiment, the present invention provide a method of surface functionalization of exosomes using the PEI-LA as disclosed herein. This method involves electrostatic surface functionalization, enhancing exosome stability, biocompatibility, and targeting accuracy under physiological conditions. The surface functionalization forms a stable, reproducible vesicle complex in aqueous environments, resistant to aggregation and proteolytic degradation. This approach enhances bioavailability, targeting precision, and therapeutic stability of the vesicles.

[0065] In a preferred embodiment, the present invention provides a method of preparing the hepatocyte-targeted exosome comprising the steps of: d) providing stem cells-derived exosomes; and e) incubating the stem cells-derived exosomes with the PEI-LA for up to 5 minutes or greater (overnight 4 degree C) in presence of NaCl solution or basal culture media to obtain the hepatocyte-targeted exosome; and f) concentrating the hepatocyte-targeted exosome by centrifugation using ultrafdters.

[0066] In an embodiment of the present invention, the stem cells-derived exosomes and the PEI-LA are used in a ratio of 1 to 2: 1 to 2, respectively. Preferably, the ratio of stem cells- derived exosomes and the PEI-LA is 2: 1.

[0067] In an embodiment of the present invention, the stem cells are selected from but not limited to bone marrow-derived mesenchymal stem cells (BMSCs), Adipose derived Stem Cells - ADSCs, Tissue-specific eg liver stem cells), Perinatal stem cells ( eg- Human umbilical cord-derived stem cells- HuMSCs), Embryonic stem cells, and the like.

[0068] In an embodiment of the present invention, the NaCl solution is used in a concentration of 1 to 1000 mM. For example, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mM. Preferably, 100 mM.

[0069] The polycationic-galactose modification of exosomes is achieved within 10-15 minutes of incubation at room temperature in a physiological relevant environment, making the process remarkably faster than existing methods. This rapid functionalization allows the formulation to be prepared immediately before administration, enhancing its practical usability and clinical relevance, especially in urgent therapeutic settings. In some embodiments, the polycationic-galactose modification of exosomes is achieved at room temperature with continuous agitation -mild vortexing at slower speeds or can be kept for overnight incubation at 4°C mild agitation

[0070] In an embodiment of the present invention, the hepatocyte-targeted exosome is formulated to precisely interact with hepatocytes, demonstrating superior targeting capabilities compared to unmodified exosomes. Characterization studies confirmed that these modifications result in a stable, reproducible nanostructure with optimal particle size, enhancing internalization into liver cells. Functional assays conducted in liver carcinoma and primary human hepatocyte models demonstrated significantly increased uptake and therapeutic efficacy of the modified exosomes.

[0071] In yet another embodiment, the present invention provides a drug-delivery system for targeted delivery to hepatocytes comprising the hepatocyte -targeted exosome as disclosed herein and a hepatoprotective drug. This is an efficient drug -delivery system where it can be directly used or can be used as cargo delivery system (n-acetyl cysteine or anti-cancer drugs, etc.) for preventing or treating the liver-related diseases.

[0072] In some embodiments, the hepatoprotective drug is selected from but not limited to the hepatocyte-targeted exosome itself or N-acetylcysteine (NAC), Glutathione, Ursodeoxycholic acid (UDCA), Ornithine aspartate, Silymarin, Curcumin, Green Tea Extract, reseveratol, and the like.

[0073] In yet another embodiment of the present invention, the hepatocyte-targeted exosome can serve as a testbed for high-end hepatocytes targeted therapies to be subjected intravenously during emergency situations or for protecting the hepatocytes before administration of hepatotoxic drugs such as Acetaminophen, Allopurinol, Isoniazid, Amoxicillin clavulanate, and the like causing idiosyncratic drug induced liver injury.

[0074] In a further embodiment, the present invention provides a pharmaceutical or hepatoprotective composition comprising the hepatocyte-targeted exosome as disclosed herein. In some embodiments, the hepatoprotective composition may be used as a nutraceutical, pharmaceutical, and the like.

[0075] In some embodiments, the hepatocyte-targeted exosome may harbour any additional active agent selected from but not limited to a prophylactic, therapeutic, or diagnostic agent that are useful in medical or veterinary applications. For example group formed by antiacids, agents against peptic ulcers and gastroesophageal reflux disease, antispasmodics, analgesics, anticholinergic drugs, propulsive drugs, antiemetics, antinausea drugs, agents for biliary therapy, agents for hepatic therapy, lipotropics, laxatives, antidiarrhetics, intestinal adsorbents, antipropulsives, anti-inflammatory drugs, active ingredients against obesity, digestive agents, enzymes, hypoglycemic drugs, insulin, vitamins, proteins, minerals, anabolic steroids, antithrombotic agents, antifibrinolytics,haemostatic agents, antiarrhythmic agents, cardiac stimulants, cardiac glycosides, vasodilators, antiadrenergic agents, antihypertensive drugs, diuretics, potassium-saving agents, antihemorrhoidals, antivaricose therapy agents, capillary stabilizing agents, agents which act on the renin-angiotensin system, beta-blockers, selective calcium-channel blockers, non-selective calcium-channel blockers, ACE inhibitors, angiotensin II inhibitors, modifying agents of lipids, antifungals, healing agents, antipruritics, antihistamines, anesthetics, antipsoriatics, chemotherapy drugs, corticosteroids, antiseptics, disinfectants, anti-acne agents, products for gynecological use, oxytocics, anticonceptives, androgen, estrogen, progestagen, gonadotropins, ovulation stimulants, antiandrogens, products for urological use, antispasmodics, drugs used in benign prostatic hypertrophy, hormones, hormone antagonists, antibiotics, tetracyclines, anphenicols, beta-lactam antibacterials, penicillin, sulfonamides, trimethoprim, macrolides, lincosamides, streptogramins, antibacterial aminoglycosides, antibacterial quinolones, antivirals, immune serum, immunoglobulins, antineoplastic agents, immunomodulatory agents, alkylation agents, antimetabolites, plant alkaloids, cytotoxic antibiotics, immunosuppressive agents, drugs for disorders of the musculoskeletal system, antirheumatics, muscle relaxant agents, agents which affect bone structure and mineralization, drugs which act on the nervous system, general anesthetics, local anesthetics, opioids, antimigraine agents, anticonvulsants, anticholinergic agents, dopaminergics, antipsychotics, anxiolytics, hypnotics, sedatives, antidepressants, psychostimulants, antidementia drugs, parasympathomimetics, drugs used in addictive disorders, anti-vertigo agents, antiparasitic agents, insecticides, insect repellants, nasal decongestants, mucolytic agents, cough suppressants, ophthalmic active ingredients, otological active ingredients, antiglaucoma drugs, miotics, mydriatics, cycloplegics and / or mixtures thereof.

[0076] In an embodiment, the pharmaceutical or hepatoprotective composition of the present invention may further one or more pharmaceutically acceptable excipients selected from but not limited to diluents, disintegrants, fdlers, bulking agents, vehicles, pH adjusting agents, stabilizers, viscosity enhancers / thickeners / gelling agents, anti-oxidants, binders, buffers, lubricants, antiadherants, coating agents, preservatives, emulsifiers, suspending agents, release controlling agents, polymers, colorants, flavoring agents, plasticizers, solvents, preservatives, glidants, and chelating agents; used either alone or in combination.

[0077] In one embodiment of the present invention, the pharmaceutical or hepatoprotective composition as disclosed herein is formulated as a parenteral formulation suitable for intravenous, intramuscular, intrahepatic, and subcutaneous routes ofadministrations. In some embodiments, the administration can be effected by hepatic perfusion.

[0078] In some embodiments of the present invention, the pharmaceutical or hepatoprotective composition as disclosed herein is administered to a subject in need thereof once in a therapeutically effective dose. In some embodiments, the pharmaceutical composition as disclosed herein is administered to a subject in need thereof more than once.

[0079] In some embodiments of the present invention, the pharmaceutical or hepatoprotective composition as disclosed herein may also be formulated into suitable dosage forms selected from a group comprising of powder, paste, tablets, syrups, infusions, capsules, and the like.

[0080] In an embodiment of the present invention, the pharmaceutical or hepatoprotective composition as disclosed herein may also be formulated in other dosage forms, including but not limited to drops, liquids, suspensions, semi-solids, solutions, emulsions, ointments and the like.

[0081] In an embodiment of the present invention, the pharmaceutical or hepatoprotective composition as disclosed herein can be used for treating liver diseases, so as to slow or reverse the patient's progression to liver failure. In this context, “liver disease” denotes a condition where liver damage and inflammation threatens to progress to a fatal loss of liver function and / or regenerative capacity. Thus, “liver disease” as used in this description encompasses hepatitis, where inflammation causes damage to liver cells and liver function, whether caused by any virus (viral hepatitis), by a liver toxin (e.g., alcoholic hepatitis), or by autoimmunity (autoimmune hepatitis). Also illustrative of “liver disease” in this description are (A) fatty liver disease (hepatic stetosis), a condition where large vacuoles of triglyceride fat accumulate in liver cells, and (B) non-alcoholic fatty liver disease, which subsumes a spectrum of disease associated with obesity and metabolic syndrome, where either (A) or (B) threatens liver damage so severe as to cause a fatal loss of liver function and / or regenerative capacity.

[0082] In an embodiment, the present disclosure provides a method of treatment, amelioration and prophylaxis of a liver disease in a subject by administering an effective amount of a pharmaceutical or hepatoprotective composition comprising the hepatocyte- targeted exosome as disclosed herein.

[0083] The dose to be administered daily is to be selected to suit the desired effect. If required, higher or lower daily doses can also be administered. Dosage maybe in a single dose or multiple doses through the day. The dosage may be varied depending on the severityof the condition, past medical history, time of administration, age, gender, weight, among other factors well known in medical art. A trained physician will be able to determine the required dosage.

[0084] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.EXAMPLES

[0085] The present disclosure is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.Material and methodsChemicals used in this study were obtained from reputable suppliers to ensure analytical- grade quality. Polyethylenimine (PEI, branched, Mw 25 kDa), lactobionic acid, EDC (1- Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide), N-hydroxy succinimide (NHS), Bovine Serum Albumin (BSA, Mw 66.5 kDa), Calcein AM, and PKH 26 were purchased from Sigma- Aldrich (USA). Additional chemicals were supplied by Loba Chemie. All chemicals utilized in the experiments were of analytical grade to ensure consistency and reliability of the results.

[0086] Example 1: Synthesis and characterization of lactosylated polyethyleneimine conjugates for specifically targeting hepatocytes within the liverOne pot synthesis was followed for synthesizing lactosylated polyetheneimine conjugates utilizing branched polyethyleneimine (PEI) and lactobionic acid (LA), where molar ratios of PEI: LA: EDC: NHS were 1:50: 100: 100. LA was added to milliQ water at pH 5 under constant stirring. To this solution, add EDC and immediately followed by NHS. Incubate the stirring reaction mixture for 30 minutes at room temperature. After the scheduled time, add the PEI solution dropwise. Keep the reaction mix for 48hr incubation at stirring at room temperature and subsequently, dialyze the solution for 48 hours with 3kDa membrane in distilled water. The solution was freeze dried at -80°C. Finally, the lyophilized PEI-LA was characterized for the specific parameters required. For different degrees of lactosylation, thesame amount of PEI amines was conjugated with different molar ratios of LA (lx, 2x, 3x, and 4x times of initial concentration) to achieve desired degree of lactosylation.For characterization of the derivatives, NMR and FTIR were performed to assess the introduction of galactose group from LA through amide bond formation. The presence of galactose concentration in different PEI-LA conjugates was calculated. Subsequently, the cytotoxicity and effect on RBCs was also assessed.Results: The 'H NMR spectrum of PEI shows chemical shift of methylene protons (CEE CEE NH) in the range of 2.4-3.5 ppm. After conjugation of PEI with LA, new peaks with chemical shift values of 3.2- 3.5 ppm were observed in spectra of PEI-LA which can be assigned to the CH protons of LA. FTIR analysis showed the absorption bands corresponding to the hydroxyl and NH groups of PEI have been shifted and the intensities were significantly changed after conjugation of LA with PEL The FTIR spectrum of PEI displayed a vibration band corresponding to primary amine (N-H) at 1496 cm'1. However, after conjugation of LA, the peak at 1496cm'1disappeared and a new peak at 1640 cm'1was observed in the spectra of PEI-LA which can be assigned to the newly formed amide bond. The lactosylation of PEI resulted in change in the zeta potential from +30 ± 3.52 mV to +9.5 ± 5.1 mV.The cytotoxicity of PEI and its derivative polymers was evaluated at varying concentration between Ipg / ml to lOmg / ml. For this, HepG2 cells were cultured and seeded at a density of 1x104cells per well in 96-well plate, one day before the evaluation of toxicity. Different concentrations were prepared in incomplete DMEM media and were added to the well plate for 4hr, and the cellular metabolic activity was evaluated using MTT assay. The hemocompatibility of the polymer derivatives was assessed by evaluating the percent hemolysis of RBCs after incubation with the polymer at desired concentration (100 pg / ml). Digital images are shown here in FIG. 1.

[0087] Example 2: Modification of the stem cells-derived exosomes and their characterizationThe exosomes were isolated from bone marrow-derived mesenchymal stem cells (BMSCs) as per the protocol mentioned in the previous study. The exosomes obtained were then incubated with PEI-LA2 conjugate for atleast 5 minutes in the ratio of 2: 1 in the presence of lOOmM NaCl solution or culture media supplemented with 10% FBS. The modified exosomes (Exo-PEI) were then centrifuged using ultrafilters (Amicon lOOkDa- Merck Millipore, USA) to finally concentrate and remove the free polymer from the system.For characterisation, first the protein concentration was measured for the modified and unmodified exosome sample using BCA protein assay (Pierce, Thermo Scientific, USA) andas per the concentration, the samples were processed and characterised for size, zeta potential and morphology by Dynamic Light Scanning (DLS), Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM). Immunogold TEM was followed for the expression of tetraspanin proteins, namely CD 9 and CD 81.Results: The exosomes were isolated from mesenchymal stromal cells derived from bone marrow and bicinchoninic acid (BCA) assay was used to quantify total protein concentration. As determined, the total protein concentration was 1100 ± 312 pg / mL. The freshly isolated exosomes were then incubated with PEI-LA2 conjugate (hereinafter will be referred to as modified exosomes or exo-PEI) in a ratio of 2: 1, as represented in the schematic illustration (FIG. 1A). The modified and native exosomes were analysed for their respective physicochemical properties and were compared for their changed characteristics. The DLS analysis of modified exosomes exhibited an increase in size range of 251 ,8±18.6 nm with a zeta potential of -10±2.3 mV. While native exosomes had size of around 146.7±24.2 nm with zeta potential -20.5±3.6 mV. NTA analysis showed the variation in the population of these exosomes, such that maximum population of modified exosomes lied around 238 nm against -132.3 nm for unmodified exosomes (FIG. ID). The exosome concentration was calculated to be around 1.84 x 108 particles / pg of protein. To further analyze the particle distribution, AFM and SEM analysis of both groups of exosomes revealed non-agglomerated nanoparticles, such that their transmission electron microscopy (TEM) image showed an increased size in case of modified exosomes (FIGs. 1C and E). The zoomed TEM images of the exosomes revealed the distinctive corona around the surface of the exosomes. The immunogold labelling of exosomes and modified exosomes with anti-CD9 and anti-CD 81 antibodies revealed the presence of exosomal markers, indicating that PEI-LA modification did not affect the physical properties of exosomes and their surface composition (FIG. IE).

[0088] Example 3: In-vitro and in-vivo assessment for exosomal uptake by the hepatocyte cellsFor the investigation of the uptake of modified- and unmodified-exosomes, hepatocellular carcinoma (HepG2) cell line was used. Exosomes were labeled with Calcein AM for 30 minutes at 37 °C, followed by two washes using centrifugation at 1000g for 15 minutes to eliminate any unbound dye. Subsequently, the labeled exosomes were modified with PEI- LA2 and subjected to an additional centrifugation step to remove unbound polymer. The modified- and unmodified- labelled exosomes were cultured with the HepG2 cells in serum- free medium at a final protein concentration of 20 pg / ml and incubated for 0, 15, 30, 60, 120and 180 minutes at 37 °C. The kinetics of respective uptake of exosomes in both the groups was examined by flow cytometry and confocal microscopy (LEICA SPE, Germany).To investigate the ability of modified exosomes to populate the liver in the pre-clinical settings. Healthy SD rats were given Pkh-labelled modified and unmodified exosomes through the lateral tail vein systemic injection. After 4hrs, the animals were sacrificed and the liver was harvested, fixed and cleared in MACS solvent or cryo-sectioned to be images under confocal laser scanning microscope.Results: The flow cytometry demonstrated that modified exosomes bound to HepG2 more efficiently than unmodified exosomes. At 120 min, modified exosomes showed a maximum uptake of around -80% against -13% uptake by unmodified exosomes. To confirm whether modified exosomes were internalized by the HepG2 cells at these time points, the cells were washed after incubation at different time points. The confocal laser-scanning microscopic analysis also revealed that there were low levels of interaction of HepG2 cells in the case of unmodified exosomes. In contrast, modified exosomes showed maximum internalization when confirmed through orthogonal sectioning. These findings confirmed the targeting and internalization ability of the ASGPR-specific exosomes. The in-vivo liver section imaging revealed the uniformly biodistributed modified exosomes when compared to the unmodified exosomes (FIG. 3A-3D).

[0089] Example 4: Hepatoprotective efficacy of modified exosomes in in-vivo acute liver injury modelThe experiments were performed as per the previously developed protocols with some modifications. Briefly, all the rats were first fasted for 24 hours before model development to generate susceptibility towards acetaminophen (APAP) toxicity due to decreased basal liver GSH levels. Then, acute liver injury was induced in all the rats (except the control group) through a single overdose of APAP. Rats received 3.5g / kg of APAP injection intraperitoneally in lOml / kg normal saline. Control animals were injected with a single dose of the same volume normal saline. Exosomes and modified exosomes were injected through tail vein 3 hours prior to acetaminophen administration in their respective groups. After 48 hours of APAP injury the rats were sacrificed to obtain their blood and organs for further processing. For biochemical analysis, the liver function tests were performed: SGOT, SGPT, Total bilirubin, ALP and INR. For histological analysis, the fixed liver tissues were paraffin- embedded and sectioned for hematoxylin and eosin staining. Histology images were acquired through microscope slide scanners (Morphle labs) while the immunohistochemistry was done using confocal microscopy (FIG. 4A-4B).Results: The hepatoprotective effect of exosomes (Exo) and modified exosomes (Exo-PEI) were injected into the rat tail vein before introducing acetaminophen-induced acute liver injury. The liver function test levels i.e. SGOT, SGPT, total bilirubin and ALP of the modified exosomes group, were found to be significantly similar to the healthy liver group. Following the administration of exosomes, notable histological alterations were detected in comparison to the healthy and acetaminophen (APAP) (PBS only) groups (FIG. 2A). Liver samples from the PBS group exhibited extensive immune cell infiltration, necrosis, sinusoidal congestion, and cellular swelling (FIG. 2A). In contrast, no significant histological changes were observed in the healthy group. Notably, hepatocyte necrosis and sinusoidal congestion were substantially reduced in both the modified exosome (Exo-PEI+APAP) and exosome (Exo+APAP) groups, with the modified exosome group showing an absence of immune cell infiltration. The expression levels of apoptosis marker, caspase 3 were analysed through confocal imaging (4C), it was found APAP group showed higher levels of caspase 3 expession in comparison to both the exosome groups. The expression levels of caspase 3 were relatively lowest in the modified exosome group. Overall, these results suggest that systemic administration of hepatocyte specific exosome nanocomplexes can efficiently prevent acute hepatic injury. This formulation can serve as a potential hepatoprotective agents against drug-induced liver injury during long-term drug therapies.

[0090] Example 5: Therapeutic efficacy of the modified exosomes in an acute-on- chronic liver injury rat modelThe experiments were performed as per the previously developed protocols with some modifications. Briefly, all the rats were divided into two groups: healthy (n=6) and CC14 treated (n=24), where healthy rats received I.P. injections of 2ml / kg of PBS in 40% olive oil. The CC14 treated rats received I.P. injections of 2ml / kg of CC14 in 40% olive oil for 6 weeks. After the development of chronic liver injury for 6 weeks, APAP -induced acute-on-chronic liver injury model was generated. For this, all the rats were first fasted for 24 hours before APAP toxicity. Then, the rats (except the healthy group) were fed a single overdose of acetaminophen (APAP) (3.5g / kg) mixed with guar gum. Control animals were injected with a single dose of the same volume normal saline. Exosomes and modified exosomes were injected through tail vein 24 hours after acetaminophen administration in their respective groups. After 7days of APAP injury the rats were sacrificed to obtain their blood and organs for further processing. For biochemical analysis, the liver function tests were performed: SGOT, SGPT, Total bilirubin, ALP and INR. For histological analysis, the fixed liver tissueswere paraffin embedded and sectioned for hematoxylin and eosin staining. Histology images were acquired through microscope slide scanners.Results: For developing the acute-on-chronic liver failure (ACLF) model, animals were first induced with repetitive CC14 chronic insults for 8 weeks. Once the chronic liver injury was observed, the animals were twice overdosed with acetaminophen (APAP), to create acute liver injury. After 24 hours of this acute-on-chronic injury, histological examination of the sacrificed rat liver revealed a drastic change in the appearance of the liver, with clear signs of inflammation and tubular necrosis (FIG. 5A). Altogether, the animals showed the characterstics biochemical and histological features of ACLF, similar to humans. Therapeutic effect of exosomes (Exo) and modified exosomes (Exo-PEI) were injected into the rat tail vein 24 hours after acetaminophen-induced acute -on-chronic liver injury. The liver function test for SGOT, SGPT, total bilirubin, albumin, and ALP were conducted at day2, day 5, and day7 after the induction of APAP injury. The blood parameters revealed a significant difference between the APAP group and the exosome (Exo+APAP) and modified exosome (Exo-PEI+APAP) groups, such that the levels were relatively lower in the modified exosome group. At day 7 post-APAP injury, the animals were sacrificed and the histological assessment by HE and sirius red revealed the presence of hepatocyte necrosis and portal inflammation in APAP group (FIG. 5B and 5C). While exosome (Exo-APAP) group showed a reduction in inflammation; however, cell necrosis and fibrotic stiffness was still observed. In contrast to this, modified exosome group (Exo-PEI+APAP) minimal cell necrosis was observed, cells were seen to be progressively organised in concentric hepatic plates; however complete resolution of fibrosis near portal zones was not observed. These results implicated the role of hepatocyte-specific exosomes nanocomplexes that helped in liver regeneration and resolved fibrosis to a certain extent when compared to unmodified exosomes.

[0091] Example 6: Assessing the role of exosomes in alleviating chronic liver complications during a CCL-induced hepatic osteodystrophy rat modelThe experiments were performed as per the previously developed protocols with some modifications. Briefly, all the rats were divided into two groups: healthy (n=6) and CCI4 treated (n=60), where healthy rats received I.P. injections of 2ml / kg of PBS in 40% olive oil. The CC14 treated rats received I.P. injections of 2ml / kg of CCI4 in 40% olive oil for 8 weeks. After the development of chronic liver injury for 8 weeks, the rats were divided into 6 groups (n=10 each group) for investigating the role of exosomes for liver and bone metabolism in the hepatic dystrophy rat model. The exosomes (modified and unmodified) were injected in the two modes of administration: systemic and intra-femur administration. The groups weredivided into healthy (positive control), CCh-trcatcd (negative control), Systemic EV (Exosome administration through tail vein), Systemic MEV (modified exosome administration through tail vein), Systemic EV + Bone EV (exosome administration through tail vein and intra-femur routes), Systemic MEV + Bone EV (modified exosome and exosome administration through tail vein and intra-femur routes respectively). The exosome and modified exosome concentration was calculated to be 200 pg / ml for every injection through tail vein or / and intra-femur, where treatment was followed up once every week for 4 weeks. Simultaneously, all the groups continued to receive CCh injury once a week to represent human chronic liver injury conditions, where injury is happening during treatment itself. At the end of 12th week, all the animals were sacrificed for assessing the biochemical parameters and histological changes in the liver and bone. For biochemical analysis, the liver function tests were performed: SGOT, SGPT, Total bilirubin, ALP and INR. For histological analysis, the fixed liver tissues were paraffin embedded and sectioned for hematoxylin and eosin staining. Histology images were acquired through microscope slide scanners.Results: For developing the hepatic dystrophy (HOD) model, animals were first induced with repetitive CCh chronic insults for 8 weeks. After 8 weeks, the animals were provided with the exosome injections along with the continuous chonic (CCh) liver injury dosage. The biochemical parameters measurement at 10th and 12th week (two and four weeks of treatment) revealed that the systemic exosome administration was more efficient to regulate the liver function test parameters (FIG. 6A). Similarly, the histological analysis revealed that the chronic liver complications were comparatively more resolved in the systemic exosome treatment when compared to the intra-bone injections alone (FIG. 6B). The microCT analysis of the femur head and distal metaphysis revealed that the localised administration of the exosome within the bone marrow had increased the bone volume when compared to the control group (FIG. 6C). However, the systemic exosome treatment and the combined systemic and bone injections resulted in significant increase in the bone volume levels and were even comparable to the healthy groups. When the peak fracture point was analysed using the tensile testing machine, the groups with systemic or systemic with intra-femur injections had the highest resistance towards the 5kN load (FIG. 6D). The results implicated that the modified exosomes with targeted ability towards liver had the maximal therapeutic efficacy during systemic intervention, such that when used in combination with intra-bone injections served as a overall approach towards management of hepatic dystrophy complications. The results of this experiments revealed that the metabolic disease conditions like hepatic dystrophy where bone mineral density gets severely affected, acting on thecausative agents or the organ like liver may ameliorate the rate of regeneration and also remineralization of bone in this case.ADVANTAGES OF THE PRESENT INVENTION:

[0092] Unique Conjugation Process: The formulation of a hepatocyte -targeted polymer- exosome nanoconjugates via controlled yet rapid conjugation of PEI-LA and exosomes via electrostatic interactions is a significant advancement. Unlike traditional covalent modification methods that may alter exosomal structure or function, this non-covalent approach maintains the exosome's structural integrity and biological activity with minimal changes to its bilipid membrane, which is crucial for therapeutic efficacy.

[0093] Rapid and Efficient Functionalization: The polycationic -galactose modification of exosomes is achieved within 10-15 minutes of incubation at room temperature in a physiological relevant environment, making the process remarkably faster than existing methods. This rapid functionalization allows the formulation to be prepared immediately before administration, enhancing its practical usability and clinical relevance, especially in urgent therapeutic settings.

[0094] Enhanced Stability in Physiological Conditions: The modified exosome formulation exhibits exceptional stability, resisting aggregation and precipitation in serum fluids, which is a common challenge for conventional exosome formulations. This stability ensures consistent performance and therapeutic reliability in vivo, significantly improving its application potential in liver treatments.

[0095] Targeted Delivery Mechanism: The use of lactosylated PEI enables specific targeting to hepatocytes via the ASGPR, a strategy not previously reported with such a rapid and efficient formulation. This specificity enhances the bioavailability and therapeutic efficacy of the exosomes, distinguishing this invention from other exosome-based delivery systems.References:1. Katsuda T, Kosaka N, Takeshita F, Ochiya T. The therapeutic potential of mesenchymal stem cell-derived extracellular vesicles. Proteomics. 2013; 13(10- 11): 1637-53.2. Zhai R, Wang Y, Qi L, Williams GM, Gao B, Song G, et al. Pharmacological Mobilization of Endogenous Bone Marrow Stem Cells Promotes Liver Regeneration after Extensive Liver Resection in Rats. Sci Rep. 2018;8( 1): 1-10.3. Le Saux S, Aarrass H, Lai-Kee-Him J, Bron P, Armengaud J, Miotello G, et al. Post-production modifications of murine mesenchymal stem cell (mMSC) derived extracellular vesicles (EVs) and impact on their cellular interaction. Biomaterials. 2020;231 (November 2019): 119675. 4. Wiklander OPB, Nordin JZ, O’Loughlin A, Gustafsson Y, Corso G, Mager I, et al. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. J Extracell Vesicles. 2015;4(2015): 1— 13.

Claims

We Claim:

1. A hepatocyte-targeted exosome presenting a targeting moiety on its surface, wherein the targeting moiety is non-covalently conjugated to surface of the exosome wherein the non- covalent conjugation is electrostatic interaction, and wherein the targeting moiety is the lactosylated polyetheneimine (PEI-LA), which targets asialoglycoprotein receptors (ASGPR) on hepatocytes.

2. The hepatocyte-targeted exosome as claimed in claim 1, wherein the PEI-LA is prepared by one-pot method of synthesis comprising the steps of: a) preparing lactobionic acid (LA) solution in water at pH 5; b) adding l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) to the LA solution, and immediately followed by N-hydroxysuccinimide (NHS) to the LA solution to obtain a reaction mixture, wherein the PEI, LA, EDC, and NHS are used in a ratio of 1:50: 100: 100, respectively; c) incubating the reaction mixture for upto 30 min at room temperature; d) adding branched polyethyleneimine (PEI) dropwise to the incubated reaction mixture from step c), followed by incubating the same for up to 48 hrs to obtain PEI-LA in solution; and e) dialyzing the solution comprising the PEI-LA for upto 48 hrs with 3kDa membrane in distilled water to obtain a purified PEI-LA.

3. A method of preparing the hepatocyte-targeted exosome as claimed in anyone of claims 1-2 comprising: a. providing stem cells-derived exosomes; and b. incubating the stem cells-derived exosomes with the PEI-LA for up to 5 minutes in presence of NaCl solution or basal culture to obtain the hepatocyte-targeted exosome; and c. concentrating the hepatocyte-targeted exosome by centrifugation using ultrafilters having a molecular weight cut-off (MWCO) of approximately 100 kDa.

4. The method as claimed in claim 3, wherein the stem cells-derived exosomes and the PEI-LA are used in a ratio of 2: 1, respectively.

5. The method as claimed in claim 3, wherein the stem cells are bone marrow-derived mesenchymal stem cells (BMSCs).

6. The method as claimed in claim 3, wherein the NaCl solution is used in a concentration of lOOmM.

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