A nanoformulation for therapeutic use

Abstract

The present invention relates to a nanoformulation comprising L-leucyl-L-leucine methyl ester (LLOMe) and a polymer for wound healing, which promotes multiple molecular signaling pathways essential for faster wound healing. The nanoformulation offers faster and improved wound and tissue healing, axon regeneration, antiviral immunity, stem cell proliferation, embryonic development, and anti-aging effects.

Description

A NANOFORMULATION FOR THERAPEUTIC USE FIELD OF THE INVENTIONThe present invention relates to a nanoformulation of L-leucyl-L-leucine methyl ester (LLOMe) and a polymer for wound healing, tissue and axon regeneration, and tumour therapy. In particular, the present invention provides a nanoformulation and the method of healing wounds, promoting tissue-regeneration, axon-regeneration, and therapy against solid tumours and virus.BACKGROUND OF THE INVENTION The treatment of wounds and the promotion of tissue regeneration represent significant challenges in medicine. Wound healing is a complex physiological process that can be disrupted by various factors, including infection, diabetes, poor circulation, and age. Chronic wounds, in particular, pose a substantial burden on patients and healthcare systems, often leading to prolonged suffering, increased risk of complications, and significant economic costs. Current wound healing therapies aim to accelerate the natural healing process, reduce inflammation, prevent infection, and promote tissue closure.Traditional approaches to wound care have included basic wound cleansing, debridement, and the application of dressings. More advanced therapies have focused on providing a moist wound environment, delivering growth factors, and stimulating angiogenesis, however, agents which could increase wound healing mechanisms are missing. Challenges in Tissue and Axon Regeneration:Beyond simple wound closure, regenerative medicine aims to restore damaged or lost tissues and organs to their original structure and function. Tissue regeneration is a complex process that involves cell proliferation, differentiation, migration, and extracellular matrix remodeling. Axon regeneration, a specific form of tissue regeneration, focuses on repairing damaged nerve fibers, particularly in the context of spinal cord injury and peripheral nerve damage. Current regenerative therapies face several challenges, including:Limited Regenerative Capacity: Many tissues, particularly in adult mammals, have limited inherent regenerative capacity.Scar Formation: The wound healing process often leads to scar formation, which can impair tissue function and prevent complete regeneration.Lack of Guidance Cues: Damaged tissues may lack the necessary signaling cues to guide cell migration and differentiation, hindering the regenerative process.Immune Response: The immune system can mount an inflammatory response that inhibits tissue regeneration.Stem Cell Therapy: Transplantation of stem cells (e.g., mesenchymal stem cells, induced pluripotent stem cells) has shown promise in promoting tissue regeneration and axon regeneration. However, challenges remain in controlling stem cell differentiation and preventing tumor formation.Current cancer therapies include surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. These treatments can have significant side effects and may not be effective in all patients. There is a need for novel therapies that are more effective and less toxic.Current antiviral therapies aim to inhibit viral replication, boost the host immune response, or block viral entry into cells. However, many viruses develop resistance to antiviral drugs, and there is a need for new antiviral agents with novel mechanisms of action.Prior Art in Cancer and Antiviral Therapies:Chemotherapy: Traditional chemotherapy drugs kill rapidly dividing cells, including cancer cells. However, they can also damage healthy cells, leading to side effects such as nausea, hair loss, and fatigue.Targeted Therapy: Targeted therapies are designed to block the growth and spread of cancer by interfering with specific molecules involved in tumor growth and progression.Immunotherapy: Immunotherapy harnesses the power of the immune system to fight cancer.Antiviral Drugs: Antiviral drugs can prevent viruses from entering cells, block viral replication, or boost the host immune response.Limitations of Existing Approaches:While the above-mentioned approaches have shown some success, they often suffer from limitations such as:Limited efficacy in chronic wounds and complex tissue damage.High cost and complexity of advanced therapies.Potential for adverse effects and complications.Incomplete tissue regeneration and scar formation.Development of drug resistance in cancer and viral therapies.In recent years, there has been growing interest in developing newer nanoformulation and methods for wound healing, tissue regeneration, axon regeneration, and therapies for solid tumours and viruses and the use of newer nanoformulation for advanced wound healing, tissue regeneration, axon regeneration, and therapies for solid tumours and viruses formulations that leverage innovative technologies and therapeutic approaches to enhance the body's natural healing processes. These formulations may incorporate a variety of components, including bioactive molecules, biomaterials, nanoparticles, and advanced delivery systems, to create synergistic effects and improve therapeutic outcomes.Accordingly, keeping in view the drawbacks of the hitherto reported prior art, the inventors of the present invention realized that there exists a dire need to provide a nanoformulation and method for wound-healing and regeneration, anti-ageing, anti-tumor, and anti-viral activity.OBJECTIVES OF THE INVENTIONThe main objective of the present invention is to provide a wound healing nanoformulation effective against skin and eye wounds comprising Dipeptide esters like L-leucyl-L-leucine methyl ester in an aqueous medium..Another objective of the present invention is to provide a tissue-regenerative and neuro-regenerative nanoformulation comprising Dipeptide esters like L-leucyl-L-leucine methyl ester in an aqueous medium.Yet another objective of the present invention is to provide a nanoformulation comprising Dipeptide esters like L-leucyl-L-leucine methyl ester and its derivatives in an aqueous medium that can induce hematopoietic progenitor or stem cells in vivo.Yet another objective of the present invention is to provide a therapeutic nanoformulation for accelerating developmental growth using Dipeptide esters like L-leucyl-L-leucine methyl ester and its derivatives.Yet another objective of the present invention is to provide a therapeutic nanoformulation for solid tumours.Still another objective of the invention is to provide new pharmaceutical applications of L-leucyl-L-leucine methyl ester and its derivatives.Yet another objective of the invention is to provide newer methods of wound healing, anti-ageing, tissue-regeneration, hematopoietic progenitor and stem cells proliferation, antiviral-state induction and tumour suppression.Yet another objective of the invention is to provide a nanoformulation to induce anti-viral state in the cells for anti-viral therapeutics.Yet another objective of the invention is to provide hydrogel and nanogel formulations of L-leucyl-L-leucine methyl ester and its derivatives capable of wound healing, anti-ageing, tissue-regeneration, hematopoietic progenitor and stem cells proliferation, anti-viral therapeutics and tumour suppression.Yet another objective of the invention is to provide a nanoformulation to trigger the signalling pathways related to embryonic development, growth, differentiation, stem cell renewal and maintenance, tissue repair, and tissue regeneration selected from L-leucyl-L-leucine methyl ester and its derivatives and other lysosomotrophic agents including glycyl-L-phenylalanine 2-naphthylamide (GPN) and sphingosine and their derivatives.Yet another objective of the invention is to provide a nanoformulation to trigger the multiple signalling pathways by other lysosomotrophic agents including glycyl-L-phenylalanine 2-naphthylamide (GPN) and Sphingosine and derivative. Yet another objective of the invention is to provide wound healing nanoformulation for wounds of the skin and eyes comprising L-leucyl-L-leucine methyl ester and its derivatives.Yet another objective of the present invention is to provide nanoformulation that induce the molecular and cellular processes important for wound healing, regeneration, anti-ageing, anti-tumor, and anti-viral.SUMMARY OF THE INVENTIONAccordingly, the present invention provides a nanoformulation of L-leucyl-L-leucine methyl ester (LLOMe) and a polymer for wound-healing, tissue and axon-regeneration, tumour therapy, inducing hematopoietic progenitor or stem cells, anti-ageing and anti-viral therapeutics.In a preferred embodiment of the present invention is therefore to provide a wound healing nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives in an aqueous medium. Another embodiment of the invention is to provide wound healing nanoformulation for wounds of the skin and eyes comprising L-leucyl-L-leucine methyl ester and its derivatives in an aqueous medium and as a nanoformulation. Another aspect of the present invention is to provide a tissue-regenerative nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives in an aqueous medium.Another aspect of the present invention is to provide a neuro-regenerative nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives..Another aspect of the present invention is to provide a nanoformulation comprising Dipeptide esters like L-leucyl-L-leucine methyl ester in an aqueous medium and nanoformulation that induces hematopoietic progenitor and stem cells in vivo.Another aspect of the present invention is to provide a therapeutic nanoformulation comprising L-leucyl-L-leucine methyl ester in an aqueous medium and nanoformulation for accelerating developmental growth using Dipeptide esters like L-leucyl-L-leucine methyl ester in an aqueous medium and nanoformulation.Another aspect of the present invention is to provide a nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives for solid tumours.Another aspect of the present invention is to provide a nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives to induce anti-viral state in the cells for anti-viral treatment.Yet another aspect of the invention is to provide new pharmaceutical applications of L-leucyl-L-leucine methyl ester comprising L-leucyl-L-leucine methyl ester and its derivatives.Yet another aspect of the invention is to provide newer methods of wound healing, hematopoietic progenitor and stem cells proliferation, tissue-regeneration and tumour suppression.Yet another aspect of the invention is to provide formulations of L-leucyl-L-leucine methyl ester and its other derivatives capable of wound healing, hematopoietic progenitor and stem cells proliferation, tissue-regeneration and tumour suppression.Yet another aspect of the invention is to provide hydrogel and nanogel formulations of L-leucyl-L-leucine methyl ester capable of wound healing, anti-ageing, tissue regeneration, hematopoietic progenitor and stem cells proliferation, antiviral state induction, and tumour suppression.Another important aspect of this invention is to offer a therapeutic nanoformulation comprising syngeneic cells treated with L-leucyl-L-leucine methyl ester injected in tumor for suppressing the growth of tumor cells.Yet another aspect of invention is to provide a nanoformulation to trigger signalling pathways related to embryonic development, growth, differentiation, stem cell renewal and maintenance, tissue repair, anti-ageing and tissue regeneration selected from L-leucyl-L-leucine methyl ester and its derivatives, and other lysosomotrophic agents including glycyl-L-phenylalanine 2-naphthylamide (GPN) and sphingosine and derivatives.In a preferred embodiment, the present invention is directed towards a wound healing nanoformulation comprising L-leucyl-L-leucine methyl ester.In another preferred embodiment, the present invention is directed towards a wound healing nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives in aqueous solutions for wounds of the skin and eyes.In a preferred embodiment, the present invention is directed towards a wound healing nanoformulation comprising L-leucyl-L-leucine methyl ester hydrobromide and L-leucyl-L-leucine methyl ester hydrochloride.In a preferred embodiment, the present invention is directed towards a regenerative nanoformulation comprising dipeptide methyl esters and their derivatives selected from L-leucyl-L-leucine methyl ester, L-leucyl-L-leucine methyl ester hydrobromide and L-leucyl-L-leucine methyl ester hydro-chloride.In another embodiment, the present invention is directed towards anti-ageing and tissue and axon-regeneration.In yet another embodiment, the present invention provides the use of 0.5-10 mM L-leucyl-L-leucine methyl ester and its derivatives for wound healing, anti-ageing, tissue and axon regeneration, proliferation of hematopoietic progenitor and stem cells, anti-viral state induction and suppression of tumours.In one embodiment, the present invention provides a method for wound healing comprising administering an aqueous solution of L-leucyl-L-leucine methyl ester in the range of 0.5-10 mM.In another embodiment, the present invention provides a method for inducing neural regeneration and anti-ageing comprising administering an aqueous solution of L-leucyl-L-leucine methyl ester in the range of 0.5-10 mM.In yet another embodiment, the present invention provides a method for inducing tumour suppression comprising administering an aqueous solution of 4-8 mM L-leucyl-L-leucine methyl ester in water.In one another embodiment, the present invention provides a method for wound healing comprising administering an aqueous solution of L-leucyl-L-leucine methyl ester in the range of 4-8 mM.In another embodiment, the present invention provides a method for inducing tissue and neural regeneration comprising administering an aqueous solution of L-leucyl-L-leucine methyl ester in the range of 4-8 mM.In yet another embodiment, the present invention provides a method for inducing tissue and neural regeneration comprising administering an aqueous solution of 2-4 mM L-leucyl-L-leucine methyl ester in water.In a further embodiment, syngeneic cells treated with 4-8 mM L-leucyl-L-leucine methyl ester and its derivatives are injected in tumor for suppressing the growth of tumor cells.In another embodiment, the aqueous medium selected in the nanoformulation is selected from water.In a further embodiment, the nanoformulation of L-leucyl-L-leucine methyl ester and its derivatives of the invention are formulated as hydrogels and nanogels.BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGSFigure 1: shows the structure of L-leucyl-L-leucine methyl ester.Figure 2: shows the morphological changes that the Mouse Embryonic Fibroblasts (MEFs) cells undergo upon treatment with L-leucyl-L-leucine methyl ester (henceforth, LLOMe). The cells first round up, then detach from the surface. In 2 to 3 hours the cells start attaching back and by 6 hours cells are completely attached to the surface. These cells proliferate normally afterwards.Figure 3: illustrates the wound healing in control, 4 mM and 8 mM LLOMe treatment. The graph represents the percentage change in wound size from the initial size (Two-way ANOVA, **p<0.01, ***p<0.001, ****p<0.0001, n=15). Figure 4: shows the graph of the relative fluorescence area of injured control and LLOMe-treated mouse eyes. (****p ≤ 0.0001, mean ± SD, n=3, two-way ANOVA, Sidak multiple comparison test)Figure 5: illustrates (A) schematic depictions of the tadpole tail regeneration model and treatment regimen. (B) shows the representative images of frog tadpole tail regeneration at the 9th day from the day of amputation. (C) represents the Graph showing length of tadpole tail at different days from amputation (Two-way ANOVA, ****p<0.0001, n=15).Figure 6: illustrates the (A) Bar graph depicting axon regrowth length after axotomy after 24 h in control and 10mM LLOMe treated group. (total number of biological repeats=4, n=17-20, Students unpaired T-test, *p<0.05, ***p<0.0005). (B) representing LLOMe (10 mM) treatment paradigm for evaluating functional restoration after axotomy in day 3 (A3) animals. Bar graph showing improved recovery index after drug treatment. (total number of biological repeats=3, n=35-38, Students unpaired T-test, ***p<0.0005). (C) representing drug treatment paradigm for evaluating aging-associated functional decline. (D) bar graph showing 10mM drug application significantly improving posterior touch response in day 5 (A5) animals. N=3 independent replicates, n=20-88 number of worms tested.Figure 7: shows the (A) graph depicting 10xSTAT-GFP reporter fluorescence intensity in immunohistochemistry confocal images of Drosophila 3rd instar larval lymph gland showing JAK / STAT pathway activation. (****p ≤ 0.0001, mean ± SD, two-tailed unpaired Student’s t-test, Welch’s correction, N (number of larvae) for control is 12 and n (number of lobes) is 25 whereas N for LLOMe treated is 15 and n is 31. (B) The graphs depict blood progenitor index per lymph gland lobe or H3P-positive cells per lobe as quantified from immunohistochemistry (IHC) confocal images of larval lymph gland showing (tep4 (Tep4-Gal4, UAS-GFP, indicated as Tep4>GFP) positive core hematopoietic progenitors and phospho-histone H3 (H3P, Serine 10) positive mitotically active cells. ***p≤0.001, ****p ≤ 0.0001, mean ± SD, two-tailed unpaired Student’s t-test, Welch’s correction, N (number of larvae) and n (number of lobes). (C) The graph depicts Relish fluorescence intensity in IHC confocal images with Relish antibody in Drosophila (Tep4-Gal4, UAS-GFP) 3rd instar larva. ***p≤0.001, mean ± SD, two-tailed unpaired Student’s t-test, Welch’s correction, N (number of larvae) and n (number of lobes).Figure 8: represents the (A) schematic representation of the experimental design depicting the timeline for LLOMe treatment and monitoring the whole body growth development in tadpoles for 35 days. (B) shows the images of control or 2mM or 4mM treated tadpoles (n=3) (C) Bar graph depict the developmental growth score of control or 2mM or 4mM LLOMe treated tadpoles (n=15, Two-way ANOVA, ****p<0.0001).Figure 9: illustrates (A) Schematic of protocol used for new cell based tumor therapy. Control trypsinized MEF cells or LLOMe treated floating MEF cells were injected intratumors (9th , 11th , 13th , and 15th day) in preformed tumors and the two groups are compared for (B) tumor volume (n=8, 16 tumors, Students unpaired T-test, ****p<0.0001) for 18 days from injections of B16F10 melanoma cells and (C) Representative picture of control tumors and LLOMe treated MEF cells injected tumors (B) Tumor weight (n=16, Students unpaired T-test, ****p<0.0001).Figure 10: shows the antiviral properties of LLOMe treated cells. (A) The qRT-PCR validation of RNA-Seq data showing upregulation of interferon response genes (B) Protein level induction of interferon stimulated gene. (C) Schematic showing LLOMe exposure regimen and infection of Japanese encephalitis virus (JEV), Vesicular stomatitis virus (VSV), and SARS-CoV-2. (D) Viral load quatification using qRT-PCR analysis from uninfected and virus infected (untreated control or 4mM treated 12h or 24h recovered) MEF cells with JEV (MOI 0.5, 24h) specific primer, VSV (MOI 1, 18h) specific primer or SARS-CoV2 (MOI 0.2, 24h) specific primers to quantify the viral load. Data are mean of triplicate samples ± SEM. (****p<0.0001, one-way Anova with Tukey's multiple comparisons test)DETAILED DESCRIPTION OF THE INVENTIONThe present invention relates to a nanoformulation of L-leucyl-L-leucine methyl ester (LLOMe) and a polymer encapsulated with chitosan nanoparticles for wound healing, tissue and axon regeneration, and tumour therapy. In particular, the present invention relates to the composition and method of healing wounds, promoting tissue-regeneration, axon-regeneration and therapy against solid tumours and viruses.L-Leucyl-L-leucine methyl ester (LLOMe) is a lysosomotropic reagent. LLOMe polymerizes inside lysosomes, causing rapid but reversible damage to the lysosomal membrane. However, the inventors here established for the first time that LLOMe could induce a program in cells that mirror the programs that governs embryonic development, stem cell proliferation and maintenance, cell growth, cell differentiation, tissue repair, and regeneration. It was found that indeed LLOMe could increase wound repair, tissue regeneration, and could reduce tumor growth and viral infection. It was found that when mouse or human cells (Mouse embryonic fibroblast (MEFs), NIH3T3, BHK21, HEK293) were treated with sublethal concentration of LLOMe (Figure 1), the cells underwent morphological changes as follows. First, the cells rounded up within 5 minutes of treatment (C2 stage, please refer to Figure 2) and then detached from the surface within 30 minutes (C3). After about 2-3 hours the treated cells, which were floating, re-attached to the surface (C4) and revived back completely like normal cells (C5, C6) (Figure 2). Next, it is investigated whether this phenomenon was specific to LLOMe and its derivatives treatment or was shared by other lysosome-disrupting agents known to induce LMP. MEFs were exposed to various LMP-inducing agents such as alum, crystalline silica, siramesine, GPN (glycyl-L-phenylalanine 2-naphthylamide) and sphingosine at different concentrations. Surprisingly, even at high concentrations of alum and silica, MEFs did not detach from the surface for an extended period. Cells treated with GPN, siramesine, LLOMe, and sphingosine exhibited rounding, detachment from the surface, and membrane blebbing. The cells treated with LLOMe, GPN, and sphingosine were able to revive, while those treated with siramesine did not. In a separate experiment, floating cells at the C3 stage were collected from all these treatments, washed, and re-plated. While cells treated with LLOMe, GPN, and sphingosine recovered normally, those treated with siramesine did not. The common feature among these three agents (LLOMe, GPN and sphingosine), that they all accumulate in lysosome and cause membrane damage resulting in Ca2+ release. To understand the molecular events that govern revival of cells, genome-wide transcriptome analysis was performed from cells taken at 6 time points / stages (Figure 2). Cluster 3 was the largest (~2300 genes) and its genes were mainly induced in C4 stage (Figure 2) , a time point, when the floating cells touched the surface to start the revival process. Cluster 1 genes (~1800) are predominantly induced in the C5 stage (Figure 2) , when most of the cells have attained their initial morphology. Multiple genes of this cluster remain induced in C6 stage as well. Cluster 2 genes (~2100) have low expression in C4 stage, induced in C5 stage and highly upregulated in C6 stage. A distinct cluster of genes was induced in temporal manner in C4, C5, and C6 stage indicating that revival of cells is a well programmed event. Next, the gene ontology (GO) enrichment analysis was performed using Metascape and Reactome tools to identify pathways / processes overrepresented in the three gene clusters (1 to 3) (Figure 3C-3D) to comprehend the status of cells at each step of revival. The cluster 3 genes represent the first step of cell revival . In Metascape analysis of this cluster , multiple pathways related to embryonic development (muscle structure, connective tissue, blood vessels, and reproductive structure), differentiation (muscles, lymphocyte, myeloid, and fat cell), organ morphogenesis, anterior-posterior pattern specification, inflammatory / immune response (cytokine production and adaptive immunity) and circadian rhythm were induced. The Reactome pathway analysis of the same cluster showed that genes related to signaling pathways such as RUNX1, WNT, NOTCH, HOX, FOXO, TGF-β, AKT, p38MAPK, JNK, RHO GTPases, interferon, and insulin receptor were upregulated . These signaling modules are known to govern embryonic development, stem cell proliferation and maintenance, cell growth, cell differentiation, tissue repair, and regeneration. In addition, at this stage, the cells have higher expression of genes related to lipid metabolism (Pparα), integrated-stress response, membrane trafficking, axon guidance, extracellular matrix organization, circadian rhythm, and chromatin remodeling. The primary transcription factors identified by TRRUST software as potential regulators of this genes program include NF-κB, Sp1, GATA2, and CREBBP among others . These transcription factors play essential roles in controlling genes crucial for embryonic development, self-renewal, maintenance, and stem cell functionality. The gene signature analysis using the PaGenBase predicted top cell type related to cluster 3 is “blastocyst”, a structure formed earliest in the embryonic development of mammals. Additionally, this analysis has also linked this cluster to embryonic stem cell lines, as well as the c2c12 undifferentiated myoblast cell line. Taken together, the data indicate that the reviving cells launch a cellular and molecular program similar to embryonic cells for reinitiating a new life and LLOMe may have potential to induce developmental programs in cells.Cluster 1 genes (C5 stage) analysis showed the overrepresentation of pathways related to cholesterol, lipids, fatty acids, carbohydrates, amino acids, nucleotides, vitamins metabolism, and ATP generation . This suggests an intense metabolic activity of the cells at this stage to support the biogenesis of organelles and other metabolic processes in the reviving cells. The inflammatory responses, specifically interferon response, which is the antiviral response of host were maintained high in this stage cells as well. Furthermore, in alignment with heightened cellular trafficking and organelle dynamics during this phase of rejuvenation, multiple genes associated with ion transportation, amino acid transportation, membrane trafficking, and autophagy were stimulated, likely to facilitate diverse biochemical, enzymatic, and metabolic requirements of biosynthetic pathways. Multiple pathways related to cell cycle, cell morphology, extracellular matrix organization, chemotaxis, etc. were also induced at this stage in order to make cells ready for cell division and migration. In the C6 stage (Cluster 2), multiple pathways related to cellular biosynthetic pathways, metabolism, membrane trafficking, cellular transport, cell morphology and chemotaxis, and inflammation remained activated . The pathways that were induced during cell resuscitation were distinctly similar to those that were required for stem cell proliferation / maintenance, tissue repair, and regeneration. Indeed, it was found that LLOMe treatment dramatically induced wound healing in a mouse model, stimulated tadpole tail regeneration in a frog model and promoted axon regeneration in the Caenorhabditis elegans model.The heatmap analysis of cell differentiation pathway genes revealed a phased reduction in gene expression up to the C3 stage, followed by a gradual increase starting from the C4 stage in a programmed manner. This data indicates that LLOMe treatment evokes a de-differentiation program of cells as the cell proceeds to floating stage, and the revival program was accompanied by re-differentiation.LLOMe could enhance wound healing in mice models.An efficient wound healing is comprised of four stages, homeostasis, inflammation, proliferation, and resolution / remodeling. Healing of wounds involves a series of well-defined morphogenetic changes that resemble embryonic development. Similar molecular pathways including Wnt / β-catenin, Notch, Tgf-b, FoxO, and Hox are critical for efficient wound healing, tissue regeneration, and embryonic developmental growth. All of the above and multiple other signaling pathways including, insulin signaling, Rho GTPases, MAPK, NF-κB, cytokine / interleukin / interferon, calcium signaling, and multiple metabolic pathways that are induced during programmed revival were surprisingly similar to the pathways induced during wound healing. So, it was tested whether LLOMe treatment could enhance wound healing in mice full excision wound model. LLOMe was dissolved in water at a concentration of 4 mM and 8 mM and 20 µl of it is topically applied to the wound surface using sterile surgical cotton every day twice a day(Figure 3). Wounds were imaged and measured daily. A dramatically faster wound healing was observed in the LLOMe treatment group (Figure 3). Within one day of treatment, an average reduction in wound size was 27% in the 4 mM LLOMe and 45% in the 8 mM group compared to 3% in control (Figure 3). By the 3rd day, where 4 mM and 8 mM LLOMe treated group average wound size was reduced by 64% and by 78%, respectively compared to only a 23% reduction in the control group (Figure 3). Thus, LLOMe could robustly induce wound healing in mouse models.Wound Healing protocol:8-10 weeks old C57BL / 6J mice were housed in groups and fed standard chow diets and all efforts were made to minimize animal suffering. Hair was removed from the mice dorsum using hair removal cream. Then the site was first cleaned by soft tissue soaked with water to remove the cream as well as hair and disinfected with skin disinfectant (70% alcohol). The mice were segregated into three groups as control, 4mM and 8mM (n=8 / group). A single cage housed a single mouse so as to minimize injuries due to home cage aggression. The mice were kept under observation for a day to check for allergic reactions.Next day, mice were anesthetized with a single intraperitoneal injection of ketamine (80 mg / kg body weight) and xylazine (16 mg / kg body weight) diluted in 100 μl of PBS (phosphate buffered saline). After the mice were fully anesthetized (5-10 mins), the surgical site was disinfected with 70% ethanol and then two full-thickness (i.e., epidermis, dermis, and subcutis) wounds (n=16 wounds / group) were induced on the dorsal skin by using a sterile punch biopsy needle (6-mm diameter). After wound creation, the animal was placed into a dry and warm chamber at 37ᴼC for its recovery from anesthesia.The wounds were topically treated daily twice with 20 µL of 4 mM and 8 mM LLOMe solution for minimum 10-15min. Similarly, the control mice were treated with 20µL of saline. The mice were treated for a total of 10 days. Everyday pictures of the wound were taken by digital camera and the area was calculated by tracing the wound margins by using ImageJ software.Measurement of the Wound Area: Each day the picture of the wound as well area was measured through digital caliper by measuring the larger and minor diameter of the wound. The wound area was calculated by applying the formula: [{( larger diameter / 2) x (Smaller diameter / 2)} x π]. The percentage of wound closure was calculated as: (area of actual wound / area of original wound) x100.LLOMe accelerates healing of corneal injury in mouse modelThe experimental mice (C57BL / 6J) of 6-8 weeks were divided into 3 groups: A) Control group without injury B) Injured Control injured without LLOMe treatment C) 4 mM LLOMe treated group. The eye of each animal before surgery or the right eye served as a negative / normal control group. Briefly, the mice were anesthetized intramuscularly with a combination of Ketamine and Xylazine followed by 1–2 drops of sterile topical 0.5% Proparacaine Hydrochloride ophthalmic solution was applied to the eye in each animal to anesthetize the eye before the surgery. Corneal epithelium damage was achieved in the left eye of the mouse by gentle scraping of the corneal epithelium using a surgical scalpel No.15, and the alkali burn was made by topical, single drop application of 0.1 N sodium hydroxide (NaOH) for 30 sec and the eye was flushed with normal saline. Epithelial damage was identified by staining the eye with fluorescein sodium ophthalmic strips, a blotting paper with orange color fluorescein stain (Care Group, Vadodara, India), and illuminated with cobalt-blue light. The positively stained area of the cornea was measured using ImageJ software (NIH, USA). LLOMe was administered topically two times a day with a 6-hour gap for 7 days in the respective groups, and the follow-up continued for days 2, 5 &7, respectively. Chemical injuries to the cornea can severely impair vision. Despite medical and surgical interventions, a considerable number of these injuries continue to lead to vision loss. Corneal wound healing involves a complex interplay of various cell types, growth factors, and cytokines. Quick re-epithelialization is essential for effective healing of the cornea. The alkali-burned cornea mouse model is the most used model to study chemical injuries to the cornea. In this model, 0.1N NaOH is used for complete debridement of corneal epithelium and is confirmed by fluorescent dye staining . LLOMe was administrated topically three times a day. The results showed a significant reduction of the epithelial defect from day 2 in LLOMe-treated groups (the green-colored area indicates epithelial defect) compared to the injured control group (Figures 4). The experiment was continued until day 7, and the extent of re-epithelialization was dramatically better in LLOMe-treated groups compared to controls (Figure 4). The results indicate that LLOMe could be a potent therapeutic option for treating corneal burn injuries.LLOMe induces tadpole tail regeneration in frog models.Next, the efficacy of LLOMe was tested in a frog tadpole (Indian tree frog, Polypedates maculatus) tail regeneration model. The tails of tadpoles at Gosner stage 26 were amputated followed by treatment with different concentrations of LLOMe (4 doses in water) and the tail length was measured for several days (Figure 5A). Blastema is a mass of undifferentiated cells, which are formed at the injury site and can regenerate into an organ or an appendage. A rapid blastema formation was observed in the treated group within one day of amputation. A significantly faster tail regeneration was observed in the treated group at all the concentrations of LLOMe compared to the control (Figure 5B and 5C). Thus, data suggest that LLOMe could enhance tail regeneration in the tadpole tail regeneration model. Frog tadpole tail regeneration protocol:Foam nests of Indian tree frog, Polypedates maculates, were collected from the ponds around the campus of the Utkal University, Bhubaneswar (200 21’N 850 53’ E), Odisha, India.Tadpoles were reared by keeping them in plastic tubs (~150 mL water for single tadpole) containing conditioned water (tap water stored and aerated for 72hr). The tadpoles were fed with boiled Amaranthus leaves.Tadpoles from Gosner stages 26 (n=12) were taken for this study. The tadpoles were anesthetized with MS 222 (Tricaine methane sulphonate) (6g / L) for 1-2 minutes prior to tail amputation. The tadpoles were kept laterally on a pre-sterilized porcelain plate and the tadpoles were amputated in the middle of the tail using a sterile blade. The amputated tadpoles were subdivided in five groups and were treated with different concentrations ranging from 0.5mM, 1mM, 2mM and 4mM of LPA-1 respectively for 1hr every alternative day (3 doses: Day-1, Day-3 and Day-5). Picture and measurement was taken in every alternative day. Measurement was taken by placing the tadpole on glass petridish with water with a graph sheet underneath the glass petridish.LLOMe enhances neuronal regeneration and suppresses the ageing response in the Caenorhabditis elegans model.To substantiate the role of LLOMe in regeneration, the Caenorhabditis elegans axon regeneration model, an excellent model for studying neuronal regeneration, was utilized. An axotomy with one of the posterior lateral microtubules (PLM) neurons was performed on day 3 (A3) animals using a pulsed UV laser and the response was measured. The axotomy of PLM reduced the posterior touch response in worms, which was measured as posterior touch response index (PTRI) as described previously and methods. The regrowth length after axotomy was significantly enhanced with LLOMe treatment (Figure 6A). Also, functional restoration in terms of posterior touch response was dramatically improved after LLOMe treatment reflected in the increased recovery index (Figure 6B). These results suggest the LLOMe treatment enhances the regeneration of axons and also increases functional recovery. Posterior gentle touch response declines with ageing as shown in Figure 6D, there is a significant drop between adult day 4 (A4) and day 5 (A5) worms (Basu et al.2017, Kumar et al.2021). We utilized this condition to screen the effect of the LLOMe on the ageing-related functional decline. LLOMe was provided for 24 hours as illustrated in the schematics Figure 6C. We found 10 mM LLOMe treatment significantly improved posterior gentle touch response in A5 animals (Figure 6D). This shows the LLOMe is effective in delaying ageing-related functional decline. C. elegans axon regeneration protocolAll the experiments were done in muIS32 [Pmec7:: GFP] background. For preparing the LLOMe plate, 3 ml of OP50 overnight culture is pelleted and dissolved in 100 µl of LLOMe solution with the required concentration. On Nematode Growth Medium (NGM) plates, 30 µl of the mixture is poured and air-dried for 30 minutes. Axotomy was performed on day 3 (A3) animals. One of the PLM neuron axons is axotomized at 50-60 µm from the cell body using pulsed UV laser (Andor, Oxford Instruments) coupled to Nikon Ti2 Eclipse microscope under 100x oil objective (NA=1.4). Axotomized worms were allowed to recover for 3 hours followed by both side PTRI measurement. Then the worms are transferred to the LLOMe plate and control OP50 seeded plates. After 24 hours of axotomy, PTRI was measured. Briefly, 10 alternative anterior and posterior gentle touch was applied using eyelash and the reversal response is scored. Posterior touch response index (PTRI) was calculated based on the number of positive responses from the 10 touches. The recovery index is calculated by dividing PTRI at 24 hours to PTRI at 3 hours. Then the worms were phenotyped using Nikon Ti2 eclipse fluorescence microscope for identifying the axotomized PLM side.LLOMe increases the hematopoietic progenitor / stem cells in the Drosophila lymph glandsAll the Drosophila stocks and crosses were maintained at 25°C, in a standard cornmeal diet. The fly stocks used were Canton S, tep4Gal4, UAS-GFP, and 10XSTAT-GFP. Canton S was used as a control strain, and Canton S flies were crossed to tep4Gal4, UAS-GFP, or 10XSTAT-GFP, and the first-generation progeny larvae were used for the studies. The early third instar larvae were collected and transferred to a vial containing distilled water and starved for 2 hours. The larvae were then transferred to a vial containing the Whatman filter paper disk on which 150 µL of 8 mM final concentration of the LLOMe was added. The larvae were treated for 14 hours with LLOMe. For control 150 µL nuclease-free water (NFW) was added to the Whatman filter paper disk.LLOMe or control-treated third instar larvae were used for lymph gland (LG) dissections. The dissections were performed in phosphate buffer saline (PBS), fixed in 4% paraformaldehyde, followed by washes with PBS containing 0.3% Triton-X (PBST). The samples were then blocked in 20% normal goat serum for 20 minutes at room temperature, followed by overnight primary antibody [rabbit anti-H3P (1:200, 06-570 – EMD Millipore)]. incubation at 4°C. This was followed by PBST washes, blocking, and treatment with appropriate Alexa-Flour conjugated secondary antibody incubation for two hours at room temperature. The LGs were then mounted in a vectashield mounting medium containing DAPI (Vector Laboratories, RRID: AB_2336790).Confocal images were captured using Zeiss LSM 780. Z projection of the confocal images was used for estimating various lymph gland parameters using ImageJ / Fiji software. The Blood Progenitor Index was estimated by measuring the percentile of GFP positive area divided by the total area of the primary lobe. A freehand selection tool was used for measuring the area of the Blood Progenitor Index. For the quantification of PH3, the positive signals for the marker were manually counted using the multipoint tool. The lymph gland quantifications were done for individual primary lymph gland lobes.The Drosophila melanogaster larval lymph gland is a well-studied model for understanding hematopoietic stem and progenitor cells (HSPCs) and recapitulates several aspects of vertebrate hematopoiesis. They are the primary site of hematopoiesis containing myeloid-like progenitor / stem cells that differentiate into functional hemocytes in the circulation of pupae and adults. Multiple signaling pathways that were perturbed by LLOMe such as NF-ĸB, WNT, NOTCH, JAK-STAT, mTOR, FOXO, and Insulin-like growth factors, were shown to play critical roles in HSPCs maintenance and function. We investigated JAK / STAT pathway activity in vivo using reporter line 10XSTAT92E-eGFP, which drives GFP expression under the control of ten STAT92E binding sites. Our analysis shows that LLOMe treatment significantly increased the 10xStat-GFP reporter activity in the Drosophila larval lymph gland as compared to the control (Figure 7A). The Drosophila larval lymph gland consists of core hematopoietic progenitors that are positive for the progenitor-specific driver thioester-containing protein-4 “tep4”87. We used tep4-Gal4, UAS-GFP X wild-type larvae where the tep4 positive population can be identified using GFP expression. Third instar Drosophila larvae were treated with LLOMe (8 mM) for 14 hours. There was a pronounced increase in the tep4 positive core progenitor index per lymph gland lobe indicating an increase in the stem cell pool upon LLOMe treatment (Figure 7B). We also observed a consequent increase in overall mitotic activity as indicated by an increase in H3P-positive cells per lymph gland lobe (Figure 7B). Further, a significant increase in the expression of Relish (NF-ĸB) was evident in the lymph glands of the treated group (Figure 7C), which is consistent with its role in maintaining the pool of hematopoietic stem and progenitor cells. Taken together, the data show that LLOMe robustly induces HSPCs in the lymph glands of Drosophila. LLOMe accelerated developmental growth in frogs.Indian tree frog, Polypedates maculates tadpoles were exposed to LLOMe in water for regimen depicted in schematic (Figure 8A) and monitored for growth for 35 days. Morphometric analysis of body growth was performed, and score provided to each Gosner stages and analyzed the morphometric differences between control and LLOMe treated group (n=15) starting from day 25. The metamorphosis was much faster in LLOMe treated group as compared to control. The 2mM LLOMe treated group was atleast 2 stage and 4 mM treated group atleast 4 stages ahead of control group. At day 35, where most of the animal in 4 mM treated group has almost completed metamorphosis (froglet, Gosner stage S45), most of the animal of the 2 mM group was at Gosner stage S42 / 43 (both limbs and tapered tail) and control group were mostly in Gosner stage S40 / 41 (only hind limb) (Figure 8B and 8C). These results showed that LLOMe could accelerate developmental growth and metamorphosis in frog’s model. Intratumor injection of LLOMe treated cells suppress tumor growth in mice model. Inducing immunogenic cell death (ICD) could be an effective strategy to enhance anti-tumor immunity and tumor regression. However, a drawback is that the ICD-inducing compounds could have pleiotropic effects, and if administrated systemically, can cause extensive damage to normal body parts. To circumvent all these problems, ICD was induced in cell culture plates using LLOMe and floating cells were injected into tumors to increase tumor immunogenicity. It was found in RNA sequencing data that LLOMe induces significant inflammatory responses especially interferon response in cells. The MEFs cells in culture plates were treated with 8 mM LLOMe for 30 minutes, and the floating cells were harvested, washed, and injected into the preformed B16 tumor xenografts (n=16, for each group) (Figure 9A). To our surprise, a dramatic reduction (>60%) was observed in the growth of tumors that were injected with LLOMe treated MEFs as compared to control trypsinized MEFs (Figure 9B, 9C and 9D). This data suggests that intratumor injection of cells treated with LLOMe could reduce tumor size significantly (Figure 9D). This is a new kind of therapy wherein compound is not needed to inject but syngeneic cells treated with LLOMe could be injected in tumor for suppressing the growth. This therapeutic strategy would not have side effects.LLOMe induces antiviral state in the cells for anti-viral immunity.An interesting observation in the transcriptome analysis of reviving cells was the robust induction of the interferon (IFN) response pathway, which is one of the most effective anti-viral responses of the host . A very large number (>100) of interferon-stimulated genes (ISGs) were found to be upregulated during cell revival . Indeed, the qRT-PCR and western blot analysis with the sentinel ISGs suggested the existence of a strong anti-viral IFN system in reviving cells (Figure 10A and 10B). It was also tested whether revived cells (12 h and 24 h) were resistant to viral infection (Figure 10C). Indeed, the revived cells were markedly resistant (>10 folds) to infection from Japanese encephalitis virus (JEV), Vesicular stomatitis virus (VSV), and SARS-CoV-2 (Figure 10D).MEF cells were seeded in a 6-well plate and treated with 4mM LPA-1 for the specified time points. The cells were then infected with JEV (MOI-1) or GFP-VSV (MOI-2.5) or SARS-COV2 (MOI-1) for indicated time points. Briefly, the cells were washed with 1X PBS and infected with the virus (diluted in serum free media) for 1 hour with manual shaking at 5-minute intervals. The viral inoculum was removed and the infected cells were washed with 1X PBS to remove any unbound virus. The cells were then maintained in complete DMEM at 37ºC with 5% CO2. Total RNA was harvested at the indicated post-infection time points for qRT-PCR analysis.For the nanoformulations, MEF cells were seeded in a 6-well plate and treated with 1ml of LLOMe-chitosan nanoparticles (NPs) in DMEM (0.5 mg / ml) which were prepared as presented in EXAMPLE 9. After 1 hour and 30 minutes, the chitosan-NPs media was removed, and the cells were maintained in complete DMEM at 37ºC with 5% CO2. Images were acquired using an EVOS 2000 microscope at 10X and 40X magnifications at specified time points.The major aspect of the present invention is therefore to provide a wound healing nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives in an aqueous medium.Another aspect of the present invention is to provide a tissue-regenerative nanoformulation comprising L-leucyl-L-leucine methyl ester.Another aspect of the present invention is to provide an anti-ageing nanoformulation comprising L-leucyl-L-leucine methyl ester.Another aspect of the present invention is to provide a neuro-regenerative nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives in an aqueous medium. Another aspect of the present invention is to provide a nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives in an aqueous medium which could induce hematopoietic progenitor and stem cells in vivo.Aqueous medium in the instant invention refers to an environment where water serves as the solvent, it’s a solution in which water dissolves other substances. These solutions contain dissolved molecules and ions surrounded by water molecules.Nanoformulations and nanogels in the instant invention refer to an environment wherein the peptide and its derivatives of instant invention are mixed or encapsulated with Chitosan nanoparticles in the presence of suitable cross-linkers like sodium tripolyphosphate (Sodium TPP) in a suitable medium and a translucent nanoparticle suspension is achieved.Another aspect of the present invention is to provide a therapeutic nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives for solid tumours.Another aspect of the present invention is to provide a therapeutic nanoformulation comprising L-leucyl-L-leucine methyl ester and its derivatives for anti-viral therapy.Yet another aspect of the invention is to provide new pharmaceutical applications of L-leucyl-L-leucine methyl ester and its derivatives.Yet another aspect of the invention is to provide hydrogel formulations of L-leucyl-L-leucine methyl ester and its derivatives capable of wound healing, anti-ageing, tissue-regeneration, induction of anti-viral therapy, proliferation of hematopoietic progenitor and stem cells and tumour suppression.Another important aspect of this invention is to offer a therapeutic nanoformulation comprising syngeneic cells treated with LLOMe and its derivatives injected in tumor for suppressing the growth of tumor cells.Yet another aspect of the present invention is to provide nanoformulation to trigger signalling pathways related to embryonic development, growth, differentiation, stem cell renewal and maintenance, tissue repair, anti-ageing, and tissue regeneration selected from a group comprising L-leucyl-L-leucine methyl ester, L-Leucyl-L-Leucine methyl ester hydrobromide, L-Leucyl-L-Leucine methyl ester hydrochloride, other lysosomotrophic agents including GPN (glycyl-L-phenylalanine 2-naphthylamide) and sphingosine.In a preferred embodiment, the derivative of L-Leucyl-L-Leucine methyl ester is selected from L-Leucyl-L-Leucine methyl ester hydrobromide and L-Leucyl-L-Leucine methyl ester hydrochloride.Another aspect of the present invention is to provide a nanoformulation to induce anti-viral state in the cells for anti-viral treatment. Proteins & Peptides face some challenges, especially in applications that demand a longer retention time in the vicinity of the diseased site. These challenges could easily be addressed through making a nanogel formulation of the peptides.Accordingly, a preferred embodiment of the invention is a nanoformulation comprising L-Leucyl-L-Leucine methyl ester and its derivatives which include in addition chitosan nanoparticles, Sodium Tripolyphosphate (Sodium TPP) cross-linkers and suitable medium. Another preferred embodiment of the instant invention is a nanogel formulation comprising L-Leucyl-L-Leucine methyl ester and its derivatives which include in addition lipopolymeric molecules, eg., Phospholipids including soya- phosphotidylcholine, dipalmitoyl-phosphatidylcholine (DPPC), Distearoyl-phosphatidylcholine (DSPC), Polymers including Chitosan, Poly(lactic-co-glycolic acid) - PLGA, etc.A preferred embodiment of this invention is a nanoformulation for wound healing, anti-ageing, tissue regeneration, tumor-suppression, induction of antiviral state and proliferation of hematopoietic progenitor and stem cells comprising L-leucyl-L-leucine methyl ester or derivatives thereof encapsulated in a nanoparticle.Another preferred embodiment of this invention is a nanoformulation for wound healing, anti-ageing, tissue regeneration, tumor-suppression, induction of antiviral state and proliferation of hematopoietic progenitor and stem cells comprising L-leucyl-L-leucine methyl ester or derivatives thereof encapsulated in a nanoparticle wherein the nanoparticles are made of polymers selected from soya- phosphotidylcholine, dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Chitosan or Poly(lactic-co-glycolic acid).In another preferred embodiment, the nanoparticles of the nanoformulation for wound healing, anti-ageing, tissue regeneration, tumor-suppression, induction of antiviral state and proliferation of hematopoietic progenitor and stem cells comprising L-leucyl-L-leucine methyl ester or derivatives thereof are made of chitosan.Another preferred embodiment of this invention is a method of preparing a nanoformulation for wound healing, anti-ageing, tissue regeneration, tumor-suppression, induction of antiviral state and proliferation of hematopoietic progenitor and stem cells comprising L-leucyl-L-leucine methyl ester or derivatives thereof encapsulated in a nanoparticle, the method comprising the steps of:%2) Preparing a polymer solution by mixing polymer selected from soya- phosphotidylcholine, dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Chitosan or Poly(lactic-co-glycolic acid) with 0.2% acetic acid followed by diluting in an organic solvent;%2) adding L-leucyl-L-leucine methyl ester to the polymer mixture in step a. followed by stirring wherein the ratio of L-leucyl-L-leucine methyl ester to the polymer is 1:2 wt / wt;%2) adding of a crosslinker to the polymer - L-leucyl-L-leucine methyl ester mixture to obtain a nanoparticle suspension.In another preferred embodiment of the nanoformulation, the organic solvent is DMEM.In another preferred embodiment of the method of preparing such nanoformulation, the cross linker is sodium Tri Polyphosphate.These unique nanogels and nanoformulations have the ability to retain the peptides in their native & active form. They also facilitate sustained release of the peptides lasting for hours to days (depending on the necessity / requirements of the application). In the case of application that require active interaction of the peptides with cellular surface (receptor / non-receptor based), the nanogels and nanoformulations (especially phospholids) would enable increased interaction of the peptides with cellular surface lipid bilayers.In the case of cancer therapeutics, the nanogel peptides of the instant invention, especially chitosan based nanogel peptides, are injected locally, and these would bind with the mucoid secretions of the cancer cells and enable better bioavailability of the peptides and sustained drug delivery.EXAMPLESThe following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.EXAMPLE 1Topical application of LLOMe heals the wound faster in mice model.LLOMe could induce wound healing in full excision wound healing mice model. The C57BL / 6 mice (4-8 weeks, male and female, body weight 20–25 grams) were individually housed.  The hairs were removed, followed by anaesthesia with ketamine and xylazine, either side of midline a full-thickness skin wound (50 mm2) was excised from the dorsum of C57BL / 6 mice. Animals are divided into Saline treatment group (controls), 4 mM LLOMe treatment group and 8 mM LLOMe treatment group with 8 mice per group.LLOMe is dissolved in water at concentration of 4 mM and 8 mM respectively and 20 µl of it was applied to the wound surface using sterile surgical cotton every day twice a day. Control group wounds are topically treated with saline solution similarly. Wounds were imaged and measured daily. A dramatically faster wound healing is observed in LLOMe treatment group. A much faster wound repair was observed with in 24 hours of topical treatment of LLOMe. On first day an average reduction is wound size was 3% in control whereas it was 27% in 4 mM LLOMe and 45% in 8 mM treatment group. By the 3rd day in the control group average wound area of 49.8 mm2 was reduced to 38.4 mm2 (by 23% reduction). In the case of 4mM LLOMe treated group average wound size of 52.8 mm2 is reduced to 19.0 mm2 (by 64% reduction) and in case 8 mM LLOMe treated group average wound size of 55.8 mm2 is reduced to 12.4 mm2 (by 78% reduction). This huge difference is maintained throughout the experiment and the wound were healed significantly faster in treatment groups. This data shows that LLOMe can significantly induce wound healing in mice model. EXAMPLE 2LLOMe induced tissue regeneration in tadpole tail regeneration modelThe treatment of LLOMe in MEFs induced significant number of pathways and genes that suggest that LLOMe could induce tissue regeneration. Next, it was tested whether LLOMe could induce tissue regeneration. For this, Indian tree frog, Polypedates maculatus tadpole tail regeneration model is used. Tadpoles from Gosner stages 26 were taken for the experiment. Tadpoles were anaesthetized with Tricaine methane sulphonate (MS 222 prior) to tail amputation through the middle of the tail. Following amputation, the tadpoles were treated with vehicle or different concentration of LLOMe ranging from 0.5 mM, 1 mM, 2 mM, and 4 mM of LPA-1 respectively for 1hr every alternative day (3 doses: Day-1, Day-3, and Day-5). For both the treated and control group of amputated tadpoles, parallel non-amputated, control tadpoles were reared. The tail length measurement was performed by placing the tadpole on glass petridish with water with a graph sheet underneath the glass petridish. Concentration dependent effect of LLOMe in inducing the tadpole tail regeneration was observed. Very significant increase in regeneration capacity of tail was observed when tadpole was treated with 2 mM and 4 mM of LLOMe. EXAMPLE 3LLOMe enhances neuronal regeneration and function in Caenorhabditis elegans axon regeneration model. To substantiate the role of LLOMe in regeneration, Caenorhabditis elegans axon regeneration model, an excellent model to study neuronal regeneration, was utilized. An axotomy with one of the posterior lateral microtubules (PLM) neurons was performed on day 3 (A3) animals using pulsed UV laser and response is measured. The axotomy of PLM reduces posterior touch response in worms, which is measured as posterior touch response index (PTRI) as described previously and methods. The regrowth length after axotomy is significantly enhanced with LLOMe treatment. Also, functional restoration in terms of posterior touch response was dramatically improved after LLOMe treatment reflected in the increased recovery index. These results suggest the LLOMe treatment enhances regeneration of axon and also increases functional recovery. EXAMPLE 4LLOMe accelerated developmental growth in frogsLLOMe exposed tadpoles showed faster developmental growth and metamorphosis. Morphometric analysis of body growth was performed and score was provided to each Gosner stages and the morphometric differences between control and LLOMe treated group (n=15) were analyzed starting from day 25. The metamorphosis was much faster in LLOMe treated group as compared to control. The 2mM LLOMe treated group was atleast 2 stage and 4 mM treated group atleast 4 stages ahead of control group. At day 35, where most of the animal in 4 mM treated group has almost completed metamorphosis (froglet, Gosner stage S45), most of the animal of the 2 mM group was at Gosner stage S42 / 43 (both limbs and tapered tail) and control group were mostly in Gosner stage S40 / 41 (only hind limb). These results show that LLOMe could accelerate developmental growth and metamorphosis in frogs model. EXAMPLE 5Suppression of tumor growth in mice model by Intratumor injection of LLOMe treated cellsInducing immunogenic cell death (ICD) could be an effective strategy to enhance anti-tumor immunity and tumor regression. However, a drawback is that the ICD-inducing compounds could have pleiotropic effects, and if administrated systemically, can cause extensive damage to normal body parts. To circumvent all these problems, ICD was induced in cell culture plates and the dead floating cells were injected into tumors to increase tumor immunogenicity. It was found that LLOMe induces significant inflammatory responses in cells. MEFs cells in culture plates were treated with LLOMe for 30 minutes, and the floating cells were harvested, washed, and injected into the preformed B16 tumor xenografts (n=16, for each group). A dramatic reduction (>60%) was observed in the growth of tumors that were injected with LLOMe treated MEFs as compared to control trypsinized MEFs. This data suggests that intratumor injection of cells treated with LLOMe could reduce tumor size significantly. EXAMPLE 6LLOMe induces antiviral immunity of cellsA very large number (>100) of interferon-stimulated genes (ISGs) were found to be upregulated during cell revival. Indeed, the qRT-PCR and western blot analysis with the sentinel ISGs suggest the existence of a strong anti-viral IFN system in reviving cells. Upon testing whether revived cells were resistant to viral infection or not, it was found that the revived cells were indeed markedly resistant (>10 folds) to infection from Japanese encephalitis virus (JEV), Vesicular stomatitis virus (VSV), and SARS-CoV-2.EXAMPLE 7LLOMe accelerates healing of corneal injury in mouse modelCorneal epithelium damage was achieved in the left eye of the mouse by gentle scraping of the corneal epithelium using a surgical scalpel No.15, and the alkali burn was made by topical, single drop application of 0.1 N sodium hydroxide (NaOH) for 30 sec and the eye was flushed with normal saline. Epithelial damage was identified by staining the eye with fluorescein sodium ophthalmic strips, a blotting paper with orange color fluorescein stain (Care Group, Vadodara, India), and illuminated with cobalt-blue light. The positively stained area of the cornea was measured using ImageJ software (NIH, USA). LLOMe was administered topically two times a day with a 6-hour gap for 7 days in the respective groups, and the follow-up continued for days 2, 5 &7, respectively. The results showed a significant reduction of the epithelial defect from day 2 in LLOMe-treated groups (the green-colored area indicates epithelial defect) compared to the injured control group (Figures 5B and 5C). The experiment was continued until day 7, and the extent of re-epithelialization was dramatically better in LLOMe-treated groups compared to controls (Figures 5B and 5C). The results indicate that LLOMe could be a potent therapeutic option for treating corneal burn injuries.EXAMPLE 8LLOMe increases the hematopoietic progenitor / stem cells in the Drosophila lymph glandsThe Drosophila melanogaster larval lymph gland is a well-studied model for understanding hematopoietic stem and progenitor cells (HSPCs) and recapitulates several aspects of vertebrate hematopoiesis. They are the primary site of hematopoiesis containing myeloid-like progenitor / stem cells that differentiate into functional hemocytes in the circulation of pupae and adults. Multiple signaling pathways that were perturbed by LLOMe such as NF-ĸB, WNT, NOTCH, JAK-STAT, mTOR, FOXO, and Insulin-like growth factors, were shown to play critical roles in HSPCs maintenance and function. We investigated JAK / STAT pathway activity in vivo using reporter line 10XSTAT92E-eGFP, which drives GFP expression under the control of ten STAT92E binding sites (Figure 8A). Our analysis shows that LLOMe treatment significantly increased the 10xStat-GFP reporter activity in the Drosophila larval lymph gland as compared to the control (Figure 8B and 8C). The Drosophila larval lymph gland consists of core hematopoietic progenitors that are positive for the progenitor-specific driver thioester-containing protein-4 “tep4”87. We used tep4-Gal4, UAS-GFP X wild-type larvae where the tep4 positive population can be identified using GFP expression (Figure 8D). Third instar Drosophila larvae were treated with LLOMe (8 mM) for 14 hours (Figure 8D). There was a pronounced increase in the tep4 positive core progenitor index per lymph gland lobe indicating an increase in the stem cell pool upon LLOMe treatment (Figure 8E and 8F). We also observed a consequent increase in overall mitotic activity as indicated by an increase in H3P-positive cells per lymph gland lobe (Figure 8E and 8F). Further, a significant increase in the expression of Relish (NF-ĸB) was evident in the lymph glands of the treated group (Figure 8G-8I), which is consistent with its role in maintaining the pool of hematopoietic stem and progenitor cells. Taken together, the data show that LLOMe robustly induces HSPCs in the lymph glands of Drosophila. EXAMPLE 9Preparation of LLOMe-chitosan nanoparticles10 mg of Chitosan is combined with 0.2% acetic acid and diluted in DMEM to a final volume of 5 ml. 5 mg of peptide is incorporated into the aforementioned chitosan solution and agitated using magnetic stirring in a beaker at 750-800 rpm. Subsequently, 1mg / mL solution of Sodium TPP (crosslinker) is added dropwise while stirring to achieve a translucent nanoparticle suspension. The pH of the nanoparticles was adjusted to reach the normal physiological range, and the final volume of the suspension was made up to 10 mL using DMEM. The final peptide concentration in the nanoparticle suspension was found to be 0.5 mg / mL.ADVANTAGES OF THE INVENTION1. Therapeutic efficacy: An easy-to-use, yet highly effective nanoformulation for wound-healing, tissue and axon regeneration, anti-viral, anti-ageing and tumour suppression therapy.2. Wound healing: The nanoformulation of the present invention induces wound-healing by having anti-ageing and tissue regeneration effect.3. Antiviral: The nanoformulation can be used for induction of anti-viral state in the cells.4. Cell proliferative: The present invention provides nanoformulation for proliferation of hematopoietic progenitor and stem cells in vivo.5. Tumor suppressive: The nanoformulation of the present invention is highly effective in the suppression of tumours where compound is not needed to inject but syngeneic cells treated with LLOMe could be injected in tumor for suppressing the growth. This therapeutic strategy does not have any significant side effects.WE CLAIM:1. A nanoformulation for wound healing comprising of:-%2) L-leucyl-L-leucine methyl ester (LLOMe) and;%2) a polymer 2. The nanoformulation as claimed in claim 1, wherein the L-leucyl-L-leucine methyl ester (LLOMe) and its derivative is encapsulated in a nanoparticle.3. The nanoformulation as claimed in claim 1, wherein the derivative of L-leucyl-L-leucine methyl ester is selected from L-leucyl-L-leucine methyl hydro-bromide and L-Leucyl-L-Leucine methyl ester hydrochloride.4. The nanoformulation as claimed in claim 1, wherein the derivative is dissolved in water.5. The method of preparing a nanoformulation as claimed in claim 1, wherein the method comprising the steps of:%2) Providing:i. polymer, ii. L-leucyl-L-leucine methyl ester,iii. cross linker. %2) preparing a polymer solution by mixing polymer as obtained in step (a) with 0.2% acetic acid, then diluting the mixture in an organic solvent;%2) adding L-leucyl-L-leucine methyl ester to the polymer mixture as obtained in step (b), then stirring L-leucyl-L-leucine methyl ester to the polymer in the ratio of 1:2 wt / wt;%2) adding crosslinker to the polymer - L-leucyl-L-leucine methyl ester mixture as obtained in step (c) to obtain a nanoparticle suspension.6. The method as claimed in claim 5, wherein the crosslinker is sodium Tri Polyphosphate. 7. The nanoformulation as claimed in claim 1, wherein the nanoparticle is made of polymer selected from the group consisting of soya- phosphotidylcholine, dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Chitosan or Poly(lactic-co-glycolic acid).8. The nanoformulation as claimed in claim 1, wherein the nanoparticle is formulated as hydrogel and nanogel.9. The nanoformulation as claimed in claim 1, wherein the size of the nanoparticle is in the range of 100-150 nm.10. Use of nanoformulation as claimed in claim 1 for Tissue regeneration.11. A method of treatment for tumor suppression by administering the nanoformulation as claimed in claim 1 in the range of 4-8 mM . ABSTRACTA NANOFORMULATION FOR THERAPEUTIC USEThe present invention relates to a nanoformulation comprising L-leucyl-L-leucine methyl ester (LLOMe) and a polymer for wound healing, which promotes multiple molecular signaling pathways essential for faster wound healing. The nanoformulation offers faster and improved wound and tissue healing, axon regeneration, antiviral immunity, stem cell proliferation, embryonic development, and anti-aging effects. 

Claims

1. A nanoformulation for wound healing comprising of:-%2) L-leucyl-L-leucine methyl ester (LLOMe) and;%2) a polymer 2. The nanoformulation as claimed in claim 1, wherein the L-leucyl-L-leucine methyl ester (LLOMe) and its derivative is encapsulated in a nanoparticle.

3. The nanoformulation as claimed in claim 1, wherein the derivative of L-leucyl-L-leucine methyl ester is selected from L-leucyl-L-leucine methyl hydro-bromide and L-Leucyl-L-Leucine methyl ester hydrochloride.

4. The nanoformulation as claimed in claim 1, wherein the derivative is dissolved in water.

5. The method of preparing a nanoformulation as claimed in claim 1, wherein the method comprising the steps of:%2) Providing:i. polymer, ii. L-leucyl-L-leucine methyl ester,iii. cross linker. %2) preparing a polymer solution by mixing polymer as obtained in step (a) with 0.2% acetic acid, then diluting the mixture in an organic solvent;%2) adding L-leucyl-L-leucine methyl ester to the polymer mixture as obtained in step (b), then stirring L-leucyl-L-leucine methyl ester to the polymer in the ratio of 1:2 wt / wt;%2) adding crosslinker to the polymer - L-leucyl-L-leucine methyl ester mixture as obtained in step (c) to obtain a nanoparticle suspension.

6. The method as claimed in claim 5, wherein the crosslinker is sodium Tri Polyphosphate.

7. The nanoformulation as claimed in claim 1, wherein the nanoparticle is made of polymer selected from the group consisting of soya- phosphotidylcholine, dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Chitosan or Poly(lactic-co-glycolic acid).

8. The nanoformulation as claimed in claim 1, wherein the nanoparticle is formulated as hydrogel and nanogel.

9. The nanoformulation as claimed in claim 1, wherein the size of the nanoparticle is in the range of 100-150 nm.

10. Use of nanoformulation as claimed in claim 1 for Tissue regeneration.

11. A method of treatment for tumor suppression by administering the nanoformulation as claimed in claim 1 in the range of 4-8 mM .   

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