NANO poly (lactic-co-glycolic) acid encapsulated tinospora cordifolia and uses thereof

Abstract

This disclosure relates to a nanoparticle composition comprising a plant extract of Tinospora cordifolia encapsulated within the nanoparticle. Also described herein are methods of treating a wound and promoting wound healing by administering the composition to a subject and compositions for use in such methods.

Description

[0001] NANO POLY (LACTIC-CO-GLYCOLIC) ACID ENCAPSULATED TINOSPORA CORDIFOLIA AND USES THEREOF

[0002] BACKGROUND OF THE INVENTION

[0003] This invention relates to a nanoparticle composition comprising a plant extract of Tinospora cordifolia encapsulated within the nanoparticle. Also described herein are methods of treating a wound and promoting wound healing by administering the composition to a subject as well as compositions for use in such methods.

[0004] Wound healing or wound repair is a highly intricate and co-ordinated process in which the skin (or another organ-tissue) repairs itself after injury. In normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer) exists in steady-state equilibrium, forming a protective barrier against the external environment. Once the protective barrier is broken, the normal (physiologic) process of wound healing is immediately set in motion. The process of wound healing is executed through a series of phases including hemostasis, inflammation, proliferation and tissue remodelling, leading to the restoration of the tissue integrity. This restoration of tissue integrity can be compromised by various factors.

[0005] Medicinal plants are becoming increasingly popular in modern society for treating a variety of diseases with less toxic and enhanced therapeutic effects. Many medicinal plants have been found useful in wound healing, thus the combination of traditional and modem knowledge can produce better pharmaceuticals for wound healing.

[0006] Tinospora cordifolia (Willd.) Miers ex Hook.f & Thoms (T. cordifolia), commonly known as Guduchi, belongs to the Menispermaceae family and is a highly potent medicinal plant used in Indian traditional ayurvedic medicine. Guduchi is well-known for its cleaning, rejuvenating, and anti-inflammatory properties. T. cordifolia (TC) has been reported to have a wide range of pharmacological properties.

[0007] Wound dressings are used to promote dermal and epidermal tissue regeneration during the wound-healing process. As a result, ideal wound dressings should not only protect the wound from microbes but also efficiently absorb exudates while remaining permeable to oxygen and moisture. Hydrogel dressings can promote wound healing by strengthening autolytic necrotic tissue and providing moisture to the wounded area. They keep the wound moist, which encourages tissue regeneration through granulation and re- epithelialization. Wound dressing is frequently used to apply topical antibiotics to wounds. However, conventional antibiotic formulations such as creams, gels, ointments, etc have significant limitations such as short drug residence time and poor patient compliance, for example due to leakage, messiness, and cost. These limitations of conventional dosage forms can be circumvented by incorporating bioactive agents in polymeric wound dressings. Numerous strategies such as protective wound dressings or skin substitutes, application of growth factors, and the use of ointments containing antibiotics have been attempted to overcome the above challenges and achieve faster healing of chronic diabetic wounds. Wound dressings and topical products are used in clinical practice to establish and maintain a moist environment and to offer favourable circumstances for healing. However, these products are frequently expensive or ineffective and may cause side effects. Many such approaches suffer from limitations which are evident from the continued increase in diabetic chronic wound cases worldwide. Therefore, cost-effective, non-toxic and effective wound treatment strategies are required.

[0008] Hydrogel dressings have proven to be effective in treating chronic wounds. The characteristics of hydrogel-based dressings can be easily adjusted in comparison to other chronic wound management treatments. Biocompatibility is essential for a hydrogel to maintain tissue homeostasis by presenting an appropriate matrix without damaging the local tissue during chronic wound healing. Pluronics, also known as poloxamers, are polyethylene oxide (PEO)-based non-ionic surfactants composed of polyethylene oxidepolypropylene oxide-polyethylene oxide (PEO-PPO-PEO) triblock copolymers. The appeal of pluronic systems stems from their safety, bio-adhesiveness, stability, and ability to form gels at low concentrations at body temperature. Amphiphilic block copolymers are among the most commonly used injectable drug delivery substrates.

[0009] In addition, poly (lactic-co-glycolic acid) (PLGA) has generated tremendous attention in pharmaceutical and biomedical applications especially in controlled drug delivery due to high biocompatibility and biodegradability. Lactate activates the production of factors responsible for vasculogenesis and the recruitment of endothelial progenitor cells in wounds. Nanocarrier-loaded gels are effective in medical applications in the treatment of a variety of disorders. Nanocarriers also have major advantages as drug delivery vehicles, such as site-specific targeting, extended systemic residence time, and protection of encapsulated molecules from metabolic pathways.

[0010] PLGA nanoparticles are one of the few FDA-approved material for human treatments mainly because PLGA nanoparticles degrade by esterase enzyme, forming lactic and glycolic acids which are known for inducing angiogenesis and fibroblast division. The use of PLGA as one of the strategies to supply lactate sustainably to achieve the goal of angiogenesis and lactate activates the production of factors responsible for vasculogenesis and the recruitment of endothelial progenitor cells in wounds.

[0011] The present invention relates to a wound healing composition using a polymeric nanoparticle encapsulated Tinospora cordifolia extract and to uses thereof. SUMMARY OF THE INVENTION

[0012] The present invention relates to a hydrogel nanoparticle composition comprising a plant extract preparation of Tinospora cordifolia encapsulated within the nanoparticle. The invention further relates to methods of treating a wound and promoting wound healing by administering the hydrogel composition to a subject, as well as to compositions for use in methods of treating a wound and promoting wound healing.

[0013] According to a first aspect of the present invention there is provided for a composition comprising: a) a plurality of nanoparticles, each of which is comprised of a biodegradable or biocompatible polymer arranged in a nanoparticle structure defining an internal lumen and an external surface; and b) a plant extract of Tinospora cordifolia encapsulated within the internal lumen of the nanoparticle.

[0014] In a first embodiment of the composition, the plant extract of Tinospora cordifolia may be an organic solvent-derived extract, for example an organic solvent selected from the group consisting of ethanol, methanol, butanol dichloromethane, chloroform, acetone and / or mixtures thereof. In one embodiment, the organic solvent is ethanol and the plant extract of Tinospora cordifolia extract is an ethanol extract of Tinospora cordifolia.

[0015] According to a second embodiment of the composition of the invention, about 70% of the plant extract is partly or wholly encapsulated within the internal lumen of the nanoparticle.

[0016] In a third embodiment of the composition of the invention, the plant extract is mixed with the nanoparticles so that each of the plant extract and the nanoparticles is dispersed throughout the composition.

[0017] According to a fourth embodiment of the composition, the biodegradable or biocompatible polymer may be selected from any biodegradable or biocompatible polymer, preferably poly(lactic-co-glycolic acid) (PLGA).

[0018] In a fifth embodiment of the composition of the invention, the nanoparticles may be in the size range of from about 200 nm to about 300 nm.

[0019] According to a further embodiment of the composition of the invention, the composition may be a hydrogel composition. For example, the hydrogel composition may comprise a polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) triblock copolymer, such as a Pluronic F127 hydrogel.

[0020] In another embodiment of the composition of the present invention, the hydrogel composition may be a thermosensitive hydrogel. In one embodiment, the hydrogel composition may be in the form of a liquid at a temperature range of from about 0 °C to about 5 °C and may be in the form of a solid gel at a temperature range of from about 35 °C to about 40 °C. In a further embodiment, the composition may comprise about 14 to 30% w / w of the hydrogel in the composition. According to a further embodiment of the composition of the invention, the composition may be a pharmaceutical composition, and may further comprise additional pharmaceutically acceptable carriers, diluents and / or excipients.

[0021] According to a second aspect of the present invention there is provided for a method of treating a wound in a subject, wherein the method comprises administering a composition as described herein to the subject.

[0022] In a first embodiment of the method of treating a wound in a subject, the method may comprise topically administering the composition to the subject. For example, the composition may be comprised in a wound dressing and the method may comprise applying the wound dressing to the subject.

[0023] According to a second embodiment of the method of treating a wound in a subject, the method may comprise administering the composition to a subject by injecting the composition. For example, the composition may be comprised in an injectable formulation and the injecting may be by intradermal or subcutaneous injection.

[0024] In a third embodiment of the method of treating a wound in a subject, the wound may be selected from the group consisting of an injury to the epidermis and / or dermis, diabetic ulcer, a pressure sore, a burn, soft tissue damage, and / or hard tissue damage, including bone and / or cartilage damage.

[0025] According to a third aspect of the present invention there is provided for a composition as described herein for use in a method of treating a wound in a subject, wherein the method comprises administering a composition to the subject.

[0026] In a first embodiment of the composition for use in a method of treating a wound in a subject, the method may comprise topically administering the composition to the subject. For example, the composition may be comprised in a wound dressing and the method may comprise applying the wound dressing to the subject.

[0027] According to a second embodiment of the composition for use in a method of treating a wound in a subject, the method may comprise administering the composition to a subject by injecting the composition. For example, the composition may be comprised in an injectable formulation and the injecting may be by intradermal or subcutaneous injection.

[0028] In a third embodiment of the composition for use in a method of treating a wound in a subject, the wound may be selected from the group consisting of an injury to the epidermis and / or dermis, diabetic ulcer, a pressure sore, a burn, soft tissue damage, and / or hard tissue damage, including bone and / or cartilage damage.

[0029] BRIEF DESCRIPTION OF THE FIGURES

[0030] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures: Figure 1 : GCMS analysis of Tinospora cordifolia stem ethanolic extract.

[0031] Figure 2: Dynamic light scattering spectra of PLGA -TC NP.

[0032] Figure 3: Field emission scanning electron microscopy (FeSEM) of PLGA-TC NP.

[0033] Figure 4: Transmission electron microscopy (TEM) of PLGA-TC NP.

[0034] Figure 5: Standard curve for TC-loaded PLGA nanoparticles, providing R2= 0.9988.

[0035] Figure 6: TEM - Energy dispersive spectrometry (EDS) of PLGA-TC nanoparticles showing successful encapsulation of Tinospora cordifolia in PLGA nanoparticles.

[0036] Figure 7: In-vitro release studies PLGA-TC Np. TC-loaded PLGA nanoparticles showed a slow and sustained release with approximately 50% of the TC components released into the medium after 48h.

[0037] Figure 8: In-vitro cytotoxic studies in L929 fibroblast cell line (MTT assay), showing cell viability at (A) 24 hrs; (B) 48 hrs; and (C) 72 hrs.

[0038] Figure 9: In-vitro scratch assay image of PluronicF-127 gel, PLGA-TC gel, PF / 0.1%w / v PLGA-TC gel and PF / 0.2%w / v PLGA-gel and the control.

[0039] Figure 10: In-vitro scratch assay graph of PluronicF-127 gel, PLGA-TC gel, PF / 0.1%w / v PLGA-TC gel and PF / 0.2%w / v PLGA-gel and the control.

[0040] Figure 11 : Hemocompatibility (RBC aggregation assay): A) positive control (saline); B) pluronic gel (blank); (C) PF / PLGA-TC 0.1%; and (D) PF / PLGA-TC 0.2 %.

[0041] Figure 12: Haemolysis results of blood on incubation with positive control (Water); negative control (saline); Pluronic gel; and PF / 0.05%w / v PLGA-TC gel; PF / 0.1%w / v PLGA-TC gel; and PF / 0.2%w / v PLGA-gel.

[0042] Figure 13: Treatment effects of PF-PLGA-TC in hydrogel on the healing of fullthickness skin wounds. Representative pictures of wound surface on days 1 , 5, 10, 15, and 20.

[0043] Figure 14: Hematoxylin and Eosin (H&E) stained histological images of skin tissue samples from healed wounds treated with TC / PLGA / 0.2%gel; positive control; blank gel; and negative control.

[0044] Figure 15: Graph of percentage of wound contraction over the course of 20 days.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0047] As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

[0048] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It is, however, contemplated as a specific embodiment of the present disclosure that the term “comprising” encompasses the possibility of no further members being present, i.e., for the purpose of such an embodiment “comprising” is to be understood as having the meaning of “consisting of”.

[0049] In its narrowest sense, the invention relates to the preparation of a temperaturesensitive Pluronics F-127 hydrogel containing poly (lactic-co-glycolic) acid (PLGA) nanoparticle encapsulated Tinospora cordifolia (TC) extract for biomedical applications, especially wound healing applications. The inventors of the present invention have prepared a hydrogel formulation of thermosensitive Pluronics F127 gel containing nano PLGA encapsulated Tinospora cordifolia extract. This formulation was studied for various biomedical applications including diabetic, burn and / or chronic wound dressing material, soft tissue and hard tissue like bone and / or cartilage regeneration.

[0050] Pluronic F-127 aqueous solutions (14 to 30% w / w) exhibit thermos-reversible properties, transitioning from a low viscous transparent solution at temperatures (4 to 5°C) to a solid gel when heated to body temperature. Pluronic F-127 hydrogels meet the criteria for an ideal burn wound dressing material that cleanses the wound of tissue detritus and debris. They are topical and inexpensive anaesthetics. These hydrogels further meet the various required characteristics and properties of an optimal initial dressing in the early management of burn wounds. The combined effect of PLGA nanoparticles and plant extracts are preferred for wound healing applications to induce faster healing and minimize scar formation.

[0051] The critical factors that influence the functional performance of the nanoparticle are particle size, morphology, surface charge, and physical state. PLGA nanoparticles were synthesized in the present study using a single nanoemulsion solvent evaporation method. PVA is a biodegradable and biocompatible synthetic biopolymer that was used as a stabilizer for PLGA nanoparticles. Using a single nanoemulsion solvent evaporation method and PVA as a stabilizer or surfactant for nanoparticle synthesis, the inventors were able to obtain nanoparticles with sizes ranging from 200 to 300 nm and an encapsulation efficiency of 70%.

[0052] The prepared nanoparticles were incorporated into a Pluronic F-127 hydrogel which acted as a carrier for transdermal delivery of phytoconstituents due to its high solubilizing ability and permeation enhancing properties. An in-situ gelling system used in an open wound or burned skin should be viscous as a free-flowing liquid to allow for easy and accurate topical administration to the injured site. At body temperature, the gel formed after phase transition should be strong enough to remain on the site for an extended time. Drug loaded in a liquid state enables the consistent diffusion of the bioactive agents in F-127 hydrogels and sol-gel transition occurs at body temperature thus providing a controlled drug release. This creates a platform for the loaded drug's continued and sustained release, which improves bioavailability, decreases systemic absorption, and reduces the need for frequent administration, resulting in better patient compliance.

[0053] The present invention provides for the use of a temperature-sensitive hydrogel containing Poly (lactic-co-glycolic) acid (PLGA) nanoparticles encapsulating Tinospora cordifolia (TC) extracts for different biomedical applications. The compositions are suitable for various biomedical applications, including treating diabetic wounds. The compositions of the present invention may be biocompatible, biodegradable and / or bioactive.

[0054] As used herein, the terms “improve”, “ameliorate”, and “heal” are used interchangeably and refer to when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and / or frequency.

[0055] As used herein, the term “treatment” or “treat” refers to any administration of the compositions herein to partially or completely alleviate, ameliorate, relieve, improve, delay onset of, reduce severity of, and / or reduce frequency, incidence or severity of one or more symptoms and / or features of a particular disorder, and / or condition. Such treatment may be of a subject who does not exhibit signs of the relevant condition or of a subject who exhibits only early signs of the condition. Alternatively, or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant condition. For example, a subject may be suffering from diabetes, and may have an early sign of a diabetic ulcer.

[0056] The term “encapsulated” is used herein to refer to substances that are completely surrounded by another material.

[0057] As used herein, the term “thermosensitive” relates to a material or substance that is sensitive to heat in one or more ways. The term “biocompatible”, as used herein, refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue e.g., in vivo. In some embodiments, materials are “biocompatible” if they are not toxic to cells. In some embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to approximately 20% cell death, and / or their administration in vivo does not induce significant inflammation or other such adverse effects.

[0058] As used herein, the term “biodegradable” refers to materials that, when introduced into cells, are broken down (e.g., by cellular machinery, such as by enzymatic degradation, by hydrolysis, and / or by combinations thereof) into components that cells can either reuse or dispose of without significant toxic effects on the cells. In certain embodiments, components generated by breakdown of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, biodegradable polymer materials break down into their component monomers. In some embodiments, breakdown of biodegradable polymer materials involves hydrolysis of ester bonds. Alternatively, or additionally, in some embodiments, breakdown of biodegradable polymer materials involves cleavage of urethane linkages. Exemplary biodegradable polymers include, for example, polymers of hydroxy acids such as lactic acid and glycolic acid, including but not limited to poly(hydroxyl acids), poly(lactic acid)(PLA), poly(glycolic acid)(PGA), poly(lactic-co-glycolic acid)(PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates, poly(lactide-co- caprolactone), blends and copolymers thereof. Many naturally occurring polymers are also biodegradable, including, for example, proteins such as albumin, collagen, gelatin and prolamines, for example, zein, and polysaccharides such as alginate, cellulose derivatives and polyhydroxyalkanoates, for example, polyhydroxybutyrate blends and copolymers thereof. Those of ordinary skill in the art will appreciate or be able to determine when such polymers are biocompatible and / or biodegradable derivatives thereof.

[0059] As used herein, the phrase “bioactive” refers to a substance that has activity in a biological system (e.g., in a cell, in a culture of cells, in a tissue, in an organism etc.). For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. It will be appreciated by those skilled in the art that often only a portion or fragment of a biologically active substance is required (e.g., is necessary and sufficient) for the activity to be present; in such circumstances, that portion or fragment is considered to be a “bioactive” portion or fragment. In some embodiments of the inventions, the bioactive is an extract of Tinospora cordifolia.

[0060] As used herein, the term “nanoparticle” refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, for example, the nanoparticle of the invention may be in the size range of from about 100 nm to about 300 nm, from about 150 nm to about 300 nm, from about 200 nm to about 300nm, or from about 250 nm to about 300 nm. In some embodiments, a nanoparticle may have a diameter of less than 100 nm. In some embodiments, nanoparticles may comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, or external surface, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define an internal lumen). In some embodiments, the micellar membrane is comprised of at least one polymer, such as for example a biocompatible and / or biodegradable polymer.

[0061] It will be understood that the composition of the invention may be in the form of a pharmaceutical composition, to be administered to a subject, to promote wound healing and tissue regeneration.

[0062] Those skilled in the art will appreciate that there are a number of methods for obtaining extracts from crude plant material. These methods include, among others, cutting, chopping, macerating and / or grinding raw plant material in at least one solvent in order to obtain a plant extract. It will also be appreciated that the plant material may be fresh material or dry plant material.

[0063] The solvent may be an organic solvent. Organic solvents typically used in the preparation of plant extracts include ethanol, methanol, butanol dichloromethane, chloroform, acetone and / or mixtures thereof. In one embodiment, the organic solvent is ethanol.

[0064] As used herein the term “crude extract” refers to a concentrated preparation of a plant extract obtained by removing secondary metabolites from the crude plant material with the aid of a suitable solvent. This may be done, for example, by submerging the crude plant material in the suitable solvent, removing the solvent and consequently evaporating all or nearly all of the solvent. As used herein the term “purified extract” refers to an extract obtained by separating the constituent parts of the crude extract from each other. By way of a non-limiting example, the constituent parts of the crude extract may be separated from one another by separating the polar constituents from the non-polar constituents. In so doing the active polar and / or non-polar constituents may thus be concentrated.

[0065] In a preferred embodiment, the prepared hydrogels can be used for tissue engineering and diabetic wound healing applications. In one embodiment, the hydrogels can be delivered through injection mode. Thus, the compositions of the invention may be formulated for intradermal or subcutaneous injection. Alternatively, or additionally, the prepared hydrogels can be used as a reversible liquid-to-gel bandage with properties that mimic many of the functions provided by the epidermis of normal skin. In some embodiments, the composition of the present invention is a composition suitable for topical use on a subject, for example impregnated in a wound dressing or transdermal patch, or for The subject may include a living animal, preferably a mammal and most preferably a human.

[0066] The composition may include a dermatologically acceptable vehicle, carrier and / or diluent. The pharmaceutical composition of the invention containing the extract may be in a form suitable for topical use. Suitable forms of the pharmaceutical composition include, for example, gels, lotions, creams, essences, emulsions, ointments, jellies or suspensions. In one embodiment of the invention, the composition may be a topical composition in the form of hydrogel. In some embodiments, the hydrogel may be contained on or impregnated into a wound dressing.

[0067] The “suitable forms” of the pharmaceutical composition may be combined with “pharmaceutically acceptable carriers” and other elements known in the art to produce the hydrogel of the present invention. The pharmaceutical composition may further be combined with other ingredients which promote absorption by the skin.

[0068] The pharmaceutical composition may be formulated by methods known to those skilled in the art. Pharmaceutically acceptable ingredients may be used. The term "pharmaceutically acceptable" refers to properties and / or substances which are acceptable for administration to a subject from a pharmacological or toxicological point of view. Further “pharmaceutically acceptable” refers to factors such as formulation, stability, patient acceptance and bioavailability which will be known to a manufacturing pharmaceutical chemist from a physical / chemical point of view.

[0069] By “pharmaceutically acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the pharmaceutical composition to a subject. The compositions of the invention may further include additives which enhance the properties of the composition. Examples of such additives include, but are not limited to rheology modifiers, suspending agents, thickeners, denaturants, humectants, solvents, emollients, emulsifiers and preservatives.

[0070] The use of the compositions entails administration of an effective amount of the pharmaceutical composition containing the extract to a subject in order to treat a condition, promote wound healing and / or promote tissue regeneration. The term “effective amount” in the context of treating a condition, promoting wound healing and / or promoting tissue regeneration refers to the administration of an amount of the composition comprising the active plant extract to an individual in need of treatment, either a single dose or several doses of the pharmaceutical composition containing the extract.

[0071] It will be appreciated by those of skill in the art that suitable dosages of the pharmaceutical composition, as well as the frequency of administration of an effective amount of the composition will be dependent on several factors. These factors include the individual components used, the formulation of the pharmaceutical composition, the condition being treated, the severity of the condition, the age, weight, health and general physical condition of the subject being treated, and other medication that the subject may be taking, and other factors as are known to those skilled in the art.

[0072] As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). In one embodiment, the subject is a human. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease or condition, such as a wound. A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0073] In the present invention, the synthesized hydrogel was non-toxic to the subject. Further, the TC-PLGA loaded Pluronic-F127 gel were favourable for fast healing in the early days of healing i.e., in stimulating re-epithelization and maturation of wounds. The present study revealed that the topical application of the hydrogel of the present invention markedly increased wound closure and wound contraction.

[0074] The following examples are offered by way of illustration and not by way of limitation.

[0075] EXAMPLE 1

[0076] Phytochemical Preparation and Analysis of Tinospora cordifolia extract

[0077] Tinospora Cordifolia (TC) was found to contain active compounds such as alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds, and polysaccharides.

[0078] The stem of the plant was collected, shade-dried and finely powdered with a grinder and extracted using the soxhlet extraction method. About 50 g of the powdered plant material was uniformly packed into a thimble and extracted using ethanol for 24 h. The solution was filtered using Whatman filter paper and the solvent was evaporated in a rotary evaporator under reduced pressure. The crude extract was kept at kept at 4 °C in the refrigerator for further analysis.

[0079] GCMS analysis was carried out on the crude extract. The large compounds were fragmented giving rise to peaks at different m / z ratios (Figure 1 , T able 1 ). Several secondary metabolites compounds including N hexadecanoicacid (antioxidant), ethyl oleate, octadecyl ester, linoleic acid ethylester, phenol, 3, 5-bis(1 ,1 -dimethylethyl) (anti-inflammatory), Phytol (antioxidant), linolaidic acid, octadecanoic acid, octadecenoic acid and eicosanoic acid were identified. Of particular interest, the compound 9,12-Octadecadienic acid (Z,Z)-fatty acid ester showed anti-inflammatory, antibacterial activity.

[0080] Table 1 : GCMS analysis of Tinospora cordifolia stem ethanolic extract EXAMPLE 2

[0081] Synthesis and Characterization of Tinospora cordifolia extract loaded PLGA nanoparticles

[0082] Synthesis of nanoarticlesTC extract loaded PLGA (L:G 50:50) nanoparticles were prepared by the nanoemulsion solvent evaporation method. Briefly, PLGA and TC extract (20% w / w) was added dissolved in 2.5 ml ethyl acetate and obtained an organic phase. Then 4 mL of aqueous phase containing 1% (w / v) Poly vinyl alcohol (PVA, Mw31000-50000 Sigma-Aldrich) was added dropwise to the prepared organic phase. As a result, an oil-in- water nanoemulsion was formed, and it was sonicated for 5 minutes. The nanoemulsion was then poured into 60 mL of 0.1 % (w / v) Poly vinyl alcohol and stirred at room temperature for 3 to 4 h until the complete evaporation of the organic solvent. It is important to note that, this transfer step must be completed very quickly, in order to avoid PLGA NPs aggregation before being stabilized by the surfactant Poly vinyl alcohol (PVA).

[0083] Unloaded nanoparticles (PLGA NP) were prepared with the same procedure except the addition of TC extract during the preparation of formulation. The resulting suspension was centrifuged at 4000 rpm, washed twice with ultra-pure water and lyophilized. The samples were stored at -20 °C for further analysis.

[0084] Particle Size Distribution and Polydispersity Index

[0085] The particle size distribution and the polydispersity index (PDI) of the nanoparticles were measured using dynamic light scattering (DLS) Horiba SZ-100. Analysis performed at scattering angle of 90°, and a refractive index of 1 .3328 (Figure 2).

[0086] Scanning electron microscopy (FeSEM)

[0087] The morphology of the nanoparticles was analyzed using a scanning electron microscope (scanning electron microscope JEOL, Tokyo, Japan). Nanoparticles were mounted on stabs and coated with gold (20 nm thickness) using a Sputter Coater coated with a gold-palladium layer using an ion sputter (JFC-1100, JEOL Ltd, Japan) and were observed under the scanning electron microscope (Figure 3).

[0088] Transmission Electron Microscopy (TEM)

[0089] A transmission electron microscope (TEM) study was carried out to further evaluate the morphological character of the nanoparticle (TEM)(JED 2100, JEOL Ltd.). A diluted suspension of lyophilized nanoparticles was placed on a film-coated with a 200-mesh copper specimen grid for 10 mins, and the excess fluid was removed with filter paper. The coated grids were dried and examined under the TEM microscope. The samples were observed by operating at 200 kV (Figure 4).

[0090] Encapsulation Efficiency

[0091] The amount of TC extract entrapped in the PLGA nanoparticles was measured using UV spectrophotometry method. In a cooling centrifuge apparatus, the sample containing TC extract was ultracentrifuged at 4,000 rpm for 30 mins. The supernatant was analysed for absorbances at Amax of 270 nm using a UV spectrophotometer, from which the concentration of the drug in the supernatant was calculated using the standard calibration data (Figure 5). The entrapment efficiency of the TC-loaded PLGA NPs was determined by the difference between the amount used to prepare the NPS and the amount found in the supernatant. The entrapment efficiency (EE%) of the PLGA-TC NPs was calculated using the formula: EE (%) = (Total amount of drug - Amount of drug in the supernatant) / Total amount of drug X 100.

[0092] Tinospora cordifolia extract was successfully encapsulated in PLGA nanoparticles, according to the presence of elemental content in PLGA-TC nanoparticles (Figure 6, Table 2).

[0093] Table 2: Elemental content in PLGA-TC nanoparticles

[0094] TC extract loaded PLGA nanoparticles formed were shown to be nanometer size, with high encapsulation efficiency. The particles formed have a size range of 200-300 nm and an encapsulation efficiency of 70%. The FeSEM and TEM images confirmed that the nanoparticles were spherically shaped and smooth surface.

[0095] EXAMPLE 3

[0096] In-vitro drug release of nanoparticles

[0097] The in-vitro drug release study of Tinospora cordifolia extract-loaded PLGA nanoparticle formulations was studied by dialysis. Drug-loaded nanoparticles (1 mg / ml) were dispersed into a dialysis bag (MWCO 12 KDa), and the dialysis bag was then kept in a beaker containing 80 mL of pH 7.4 phosphate buffer. The beaker was placed over a magnetic stirrer, and the temperature of the assembly was maintained at 37 ± 1 °C throughout the experiment. During the experiment, rpm was maintained at 100 rpm. Samples (2 mL) were withdrawn at specific time intervals and replaced with equal amounts of fresh pH 7.4 phosphate buffer. These samples were filtered through a 0.2- m nylon membrane filter, and the collected samples were analysed using a UV-visible spectrophotometer (Figure 7).

[0098] In-vitro release of plant extract encapsulated poly (lactic-co-lactic acid) nanoparticles shows a sustained release of the drug. Approximately 50% of the TC components were released into the medium after 48 h. PLGA nanoparticles undergo biodegradation via the diffusion and erosion process and allow the herbal extract to diffuse out of the nanoparticle at a constant rate. This property not only reduced the quantity of the active (T. cordifolia) but also increased the efficacy due to prolonged sustained release.

[0099] EXAMPLE 4

[0100] Synthesis of temperature-sensitive Pluronics F-127 containing PLGA nanoparticles encapsulating Tinospora cordifolia extract

[0101] Preparation of Pluronic (F127) Solution

[0102] Pluronic F-127 aqueous solutions 20% w / w were synthesized using cold method which exhibit thermos-reversible properties, transitioning from a low viscous transparent solution at temperatures (4 to 5 °C) to a solid gel when heated to body temperature. The cold method involves the slow addition of polymer to cold water with continuous agitation. The mixture was stored at 4 °C overnight to obtain a clear solution.

[0103] Preparation of Pluronic F-127 gel containing Tinospora cordifolia extract-loaded PLGA nanoparticles

[0104] TC extract loaded PLGA nanoparticles were incorporated into Pluronics F-127 gel and tested the in-vitro and in-vivo wound healing potential. PF-127 gel (20% w / v concentration) was prepared by dissolving PF-127 powder in distilled water at 4 °C. The solution was shaken vigorously and cooled at 4 °C for 6-8 h. After liquefaction was completed, it was stored at 4 °C. TC-PLGA nanoparticles were frozen at -20 °C for 24 h. TC-PLGA nanoparticles were incorporated into pluronic F-127 by direct mixing at 4 °C for 6 h.

[0105] Gelation Temperature (Tgel) and Viscosity Measurement

[0106] 4mL of cold sample solutions were placed in a beaker (25 mL) and placed in a roomtemperature water bath. In the sample solution, a thermometer was immersed. The solution was heated at a constant rate of 200 rpm under continuous magnetic stirring. The gelation temperature was measured as the temperature at which the magnetic bar stopped moving.

[0107] Viscosity analysis was performed on the synthesized formulations using a Brookfield. Viscometer with Spindle no. 4 (Brookfield Engineering Lab., Inc., Stoughton, MA.02072, USA ) at 25 °C and a spindle speed of 20 rpm. The resulting formulations of TC-PLGA NP containing Pluronics F127 20w / v % are shown in Table 3 below.

[0108] Table 3: Formulations of TC-PLGA NP containing Pluronics F127 20 w / v %

[0109] The resulting hydrogel was determined to have a consistency as appropriate for an injectable formulation which can be delivered to the site of injury / disease by routine injectable delivery mechanisms.

[0110] EXAMPLE 5

[0111] In vitro studies using pluronic F127 gel containing PLGA nano-encapsulated Tinospora cordiofolia extract

[0112] In-vitro toxicity

[0113] Cytotoxicity of the hydrogel was evaluated by extraction method recommended by International Organization for Standardization (ISO / EN 10993-5). MTT (3-(4,5- dimethylthyazol-2-yl)-2,5-diphenyltetrazolium bromide) test was employed to assess cell proliferation. In a 96-well plate, the mouse L929 fibroblast cell line was seeded at a density of 3 x 104cells / ml and grown in DMEM medium (Sigma-Aldrich, St Louis, MO, USA). 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 1 % penicillinstreptomycin solution were added to the media to increase it. The gel was first incubated in the medium at 37 °C for 24 h. Later, syringe filters were used to filter the samples, which were then analyzed. L929 fibroblast cells were cultured overnight at 37 °C in an incubator with a 5% CO2 supply before being treated with different concentrations of gel extracts from semi-physiological media for different timelines (24, 48, and 72 h) and their cytocompatibility was assessed. The absorbance of each sample at 570 nm was measured on a microplate reader. The cell viability was calculated using the equation: Cell viability (%) = OD of sample / OD of control x 100.

[0114] In-vitro cytotoxic studies in L929 fibroblast cell line prove pluronic gel / T. cordofolia extract / PLGA nanoparticles to be cytocompatible in the light of MTT assay conducted in 24h,48h, and 72 h (Figure 8). Scratch assay

[0115] The in vitro wound healing scratch test was performed using L929 mouse fibroblast cell lines to determine the effect of the PLGA-TC PF gel on fibroblast cell migration. In each independent experiment, cells were grown in 24-well cell culture plates in DMEM medium and the test was carried out according to a previously reported protocol (Augustine et aL, 2020). Briefly, after removing the medium, a confluent layer of cells was scratched on the plate with a 100 pL pipette tip. After gently washing to remove floating cells, the samples: medium containing extracted PLGA-TC PF gel (treatment) and DMSO (negative control), were placed over the scratches, and fresh medium added, then samples were placed in a CO incubator (37 °C, 5% CO supply). Images of the plate were taken at regular intervals using an inverted microscope (Leica DMi1 ) immediately following the experiment. Images of the plates were captured with an inverted microscope (Leica DMi1 ) immediately after the experiment and after a regular time interval. In vitro wound healing (%) was determined by quantifying the cell-free scratched area using the equation: In vitro wound healing (%) = (WdO - Wdt ) / WdO x 100, wherein WdO and Wdt are the average distance between scratch boarders soon after the wounding procedure and after time ‘t’ of treatment.

[0116] In vitro scratch assay was performed to elucidate the cell migration ability of fibroblasts. The results, as shown in Figures 9-10 and Table 4, demonstrate the rapid migration of cells in the Pluronic F127 gel, PF / 0.1 % PLGA-TC gel and PF / 0.2% PLGA-TC gel formulations.

[0117] Table 4: Quantified results of the scratch assay

[0118] Blood Compatibility studies

[0119] To evaluate the hemocompatibility of the PF-PLGA-TC gel at various concentrations, blood cell aggregation, hemolysis tests were performed as previously reported. Briefly, blood samples (approximately 10 mL) from healthy volunteers were collected in tubes containing anticoagulant (3.8% sodium citrate in a 9:1 ratio). Pre-sterile Pluronic gel, PF / 0.0.05% PLGA-TC gel, PF / 0.1 % PLGA-TC gel, and PF / 0.2% PLGA-TC gel were extracted by incubating in 1 mL PBS for 12 h and the extract was collected by Sartorius Minisart® syringe filters PES 0.1 pm (Germany) and used for red blood cell (RBC) aggregation and hemolysis tests. Blood cell aggregation study

[0120] Blood was centrifuged at 700 rpm for 10 minutes to separate the RBCs. The cell fraction obtained was washed twice with saline and diluted with PBS in the ratio 1 :4. The diluted 2 ml RBCs were incubated for 20 min at 37 °C with 1 ml of each PBS extract of test samples. The negative (water) and positive (saline) controls were also incubated with the diluted RBCs and examined on mounted slides using phase-contrast microscopy (Leica, DMIRB, Germany).

[0121] There was no aggregation of cells in any of the samples on incubation. Normal saline was used as the negative control and is comparable with the results of the gels. This indicates that the morphologic features of the cells were maintained. It can be assumed that PF / PLGA-TC gel is compatible with blood. Both experiments revealed that GEL / TC / PLGA samples do not show RBC aggregation as well as hemolysis suggesting its potential in wound healing applications (Figure 1 1 ).

[0122] Hemolysis assay

[0123] For the hemolysis assay, 100pL of RBCs, with 800pL of saline, andlOOpL of each PBS extract of test samples in various concentrations (Pluronic gel, PF / 0.0.05% PLGA-TC gel, PF / 0.1% PLGA-TC gel, and PF / 0.2% PLGA-TC gel) were used as a reaction mixture. Normal saline and distilled water were used as negative and positive controls respectively. The samples were then incubated for 30 min at 37 °C followed by centrifugation at 700 rpm for 5 min. The optical density of the sample was measured at 541 nm using a UV-visible spectrophotometer (Shimadzu double beam spectrophotometer - model 1700, Japan). The percentage of hemolysis was calculated using the equation: % Hemolysis = [OD of sample - OD of negative control] / [OD of positive control - OD of negative control] x 100.

[0124] The results of the haemolysis assay are shown in Figure 12 and Table 5. Hemolysis and RBC aggregation assay show blood compatibility, as well as MTT and scratch assay in-vitro wound healing), demonstrating non-toxic and cyto-friendly nature with enhanced cell attachment and cell proliferation properties of this novel formulation. It is evidenced from in-vivo diabetic murine models that this novel formulation enhances wound closure, epithelialization, and hair follicle formation in diabetic chronic wounds. T able 5: Haemolysis results of the positive control (water), negative control (saline), pluronic gel and PF / 0.05%w / v, PLGA-TC gel, PF / 0.1%w / v PLGA-TC gel and PF / 0.2%w / v PLGA- gel

[0125] EXAMPLE 6

[0126] In vivo testing of temperature-sensitive Pluronics F-127 containing PLGA nanoparticles encapsulating Tinospora cordifolia extract

[0127] Wound healing experiments were performed using diabetes-induced male Sprague Dawley rats with an average weight of 220 g which were obtained from Kerala Veterinary and Animal Sciences University, Mannuthy, Kerala, India. Animal experiments were performed with the prior consent of the Institutional Animal Ethics Committee no. (602 / P0 / Re / S / 2002 / CPCSEA) of the Pushpagiri Institute of Medical Sciences and Research Centre, Kerala, India, in compliance with the regulations of the Committee for control and Supervision of Experiments on Animals (CPCSEA) established by the Ministry of Environment, Forests and Climate Change, Animal Welfare Division of Government of India.

[0128] Diabetes in rats was induced by the administration of streptozotocin (intraperitoneal injection, 40 mg / kg body weight, Sigma Aldrich, USA) glucose level (150-200 mg / dL).

[0129] General anaesthesia with ketamine hydrochloride (50 mg / kg) and xylazine (5 mg / kg) were administered intraperitoneally. Before creating wounds, the dorsal surface of the animals was shaved with a sterile blade while the animals were under anaesthesia. Povidone-iodine was used to sterilize the dorsal area. Four full-thickness punches were made in the skin using surgical punches (10 mm) at the center of the shaved skin. The four wounds in each animal were treated with blank (Pluronic F-127 gel), 3 treatment group PLGA-TC-PF / 0.2 % w / v gel, Positive control (Mupirocin), and Negative control (nontreated). The wounds were then covered with Tegaderm. The dressings were opened every 5th’ 10th, 15th, and 20thday until the complete healing of the wounds, was visually examined, and the diameter of wounds was measured with a scale. The images were taken using a camera (Nikon D850) (Figure 13). The wound contraction (%) was calculated using the equation: Wound healing (%) = (Initial wound area - Final wound area) / Initial wound area x 100. On day 21 , surgical samples of healed skin tissue were collected for histopathological analysis using hematoxylin and eosin (H&E) staining. The dorsal skin section containing the healed wound was resected and fixed in 10 % formalin for histological evaluation. After processing in an automated tabletop tissue processor (Leica Biosystems, Germany), it was embedded in paraffin wax. Tissue sections (4 pm thickness) were made using an automated microtome (Leica, Biosystems, Germany). All sections were stained with Harris’s hematoxylin and eosin (H&E) stain for histomorphology. After hematoxylin and eosin staining, sections were investigated for histomorphology by observing under a polarized optical microscope (Leica DM 4500 P LED) and images were captured (Figure 14).

[0130] Local tissue response to pluronic gel that serves as blank and treatment PF / 0.2%w / v PLGA-TC gel was evaluated as per the guidelines of ISO standard 10993- Part 6 (2016). The number of inflammatory cells (neutrophils, plasma cells, lymphocytes, and macrophages), the number of foreign body giant cells, the severity of necrosis, the extent of neovascularization, the extent of fibrosis, and the extent of fatty infiltration, epithelialization, and angiogenesis were all assessed in each H&E-stained section.

[0131] When evaluating the wound healing results of this study, it is observed that, Pluronics F-127 gel containing TC extract loaded PLGA nanoparticles (PLGA-TC-0.2w / v% PF gel) is more effective compared to control and the blank gel. According to histological analysis, PF gel formulations containing TC extract loaded PLGA NP accelerate the healing process by promoting re-epithelialization and angiogenesis in damaged tissues. Similarly, recovery was faster when wound diameter contraction was assessed.

[0132] The ratio of wound contraction was calculated to quantify the extent of wound healing and the results are shown in wounds treated with PLGA-0.2w / v% PF gel showed 100% wound contraction on the 21 st day of the study while 97.03% for positive control and 98.7% for blank gel (Figure 12, Table 6). The main cells involved in the healing of wounds are fibroblasts which move from the edges of the wound to the centre facilitating the synthesis of extra-cellular matrices. The wound area was significantly reduced with TC- PLGA-0.2w / v% PF gel, which showed the greatest contraction. The results show that the pluronic hydrogel containing PLGA nano-encapsulated Tinospora cordiofolia extract of the present invention had excellent wound closure observed on the 10thday, in comparison with the negative control and positive control (mupirocin).

[0133] On the 10thday of the study, the wound site treated with the TC -PLGA-0.2w / v% PF gel was fully healed and recovered with the normal dermal region leaving only a light mark (Figure 13). Table 6: Percentage of wound contraction

[0134] In the case of wound healing, 100% significant wound healing was observed on the 15thday of treatment (p=0.001282). A significant difference was found between negative control and treatment (p=0.034339). In between positive control and treatment, no significant difference was observed or only a marginal difference was observed (p=0.06801 ) (Figure 15).

[0135] Statistical analysis

[0136] Statistical analysis was carried out using R (version 4.2.2) statistical software. Initially, the mean and standard deviation was calculated and the means of the treatment as well as time (in days) and control group were subjected to ANOVA. Similarly, a pairwise comparison for both was carried out separately using two tailed paired t-test. P<0.05 was considered significant. Plots were generated using ggplot2 package of R.

[0137] ANOVA for time (in Days)

[0138] The results in Table 7 below, based on the p-value (p<0.05), show that there is a significant difference between the effect of chemical used for wound healing (row). As well as a significant difference also exist between the days of treatment (column). In other words, we fail to reject alternate hypothesis (H1).

[0139] Table 7: ANOVA for time (in Days)

[0140] ANOVA for chemical used for wound healing

[0141] Similarly, the results in Table 8 below, based on the p-value (p<0.05), show that there is a significant difference between the effect of chemical used for wound healing (columns). As well as a much significant difference (p<0.001 ) also exist between the days of treatment (rows). In other words, we fail to reject alternate hypothesis (H1). Table 8: ANOVA for chemical used for wound healing

[0142] Two-tailed paired t-test to compare the significance of time

[0143] From Table 9 below, it is clear that a positive corelation exist between the days. Stronger corelation observed in day 10 and day 20 (underlined) while less corelation was between day 0 and day 20 (bold). In both one tailed and two tailed t-test a statistical significance was observed (p<0.05), however the result for day 15 and day 20 were not significant at two tailed t-test bold italics).

[0144] Table 9: Two-tailed paired t-test to compare the significance of time

[0145] Two-tailed paired t-test to compare the significance of chemicals used for wound healing

[0146] Further, T able 10 below shows that a positive corelation exists between the chemical used for wound healing.

[0147] In one tailed t-test a statistical significance was observed (p<0.05) in case of negative control and treatment. While marginal significance exist in case of positive control and treatment and negative control and positive control.

[0148] In case of two tailed t-test a statistical significance was observed (p<0.05) only in case of negative control and treatment (bold), whereas marginal or no significance was observed for positive control treatment and negative control and positive control at two tailed t-test bold italics). Table 10: Two-tailed paired t-test to compare the significance of chemicals used for wound healing

[0149] With the promising wound healing results from the present study, it can be concluded that the PF / PLGA-TC hydrogel with enough exudate uptake capacity to ensure a suitable microenvironment for cell proliferation could be a highly favorable material for faster wound healing applications even in the case stringent diabetic wound ulcers. Pluronics F-127 also has a significant effect on wound healing which showed a synergistic effect with the nanoparticles. The use of Pluronics F-127-based gel formulation containing Tinospora cordifolia extract-loaded PLGA nanoparticles present a new perspective on wound healing and advantages compared to conventional medicinal preparations.

Claims

CLAIMS1. A composition comprising: a) a plurality of nanoparticles, each of which is comprised of a biodegradable or biocompatible polymer arranged in a nanoparticle structure defining an internal lumen and an external surface; and b) a plant extract of Tinospora cordifolia encapsulated within the internal lumen of the nanoparticle.

2. The composition of claim 1 , wherein the plant extract of Tinospora cordifolia is an organic solvent-derived extract.

3. The composition of claim 2, wherein the plant extract of Tinospora cordifolia is an ethanol extract.

4. The composition of any one of claims 1 to 3, wherein about 70% of the plant extract is partly or wholly encapsulated within the internal lumen of the nanoparticle.

5. The composition of any one of claims 1 to 4, wherein the plant extract is mixed with the nanoparticles so that each is dispersed throughout the composition.

6. The composition of any one of claims 1 to 5, wherein the biodegradable or biocompatible polymer is poly(lactic-co-glycolic acid) (PLGA).

7. The composition of any one of clams 1 to 6, wherein the nanoparticles are in the size range of from about 200 nm to about 300 nm.

8. The composition of any one of claims 1 to 7, wherein the composition is a hydrogel composition, wherein the hydrogel composition comprises a polyethylene oxidepolypropylene oxide-polyethylene oxide (PEO-PPO-PEO) triblock copolymer.

9. The composition of claim 8, wherein the hydrogel is a Pluronic F127 hydrogel.

10. The composition of claim 8 or 9, wherein the hydrogel composition is thermosensitive and / or wherein the hydrogel composition is a liquid at a temperature range of from about 0 °C to about 5 °C and is a solid gel at a temperature range of from about 35 ° to about 40 °C.1 1. The composition of any one of claims 8 to 10, wherein the composition comprises about 14 to 30% w / w of the hydrogel in the composition.

12. The composition of any one of claims 1 to 11 , wherein the composition is a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier, diluent and / or excipient.

13. The composition of any one of claims 1 to 12 for use in a method of treating a wound in a subject, the method comprising administering the composition to the subject.

14. The composition for use of claim 13, wherein the administering comprises topically administering the composition to the subject, optionally wherein the composition is comprised in a wound dressing and topically administering the composition comprises applying the wound dressing to the subject.

15. The composition for use of claim 13, wherein the administering comprises injecting the composition, optionally wherein the injecting is by intradermal or subcutaneous injection.

16. The composition for use of any one of claims 13 to 15, wherein the wound is selected from the group consisting of an injury to the epidermis and / or dermis, diabetic ulcer, a pressure sore, a burn, soft tissue damage, and / or hard tissue damage, including bone and / or cartilage damage.

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