FIELD OF THE INVENTION:

The present invention provides a novel casein manganese nanoparticle and its therapeutic angiogenesis application. More particularly, the present invention relates to simple, fast, efficient, economically cheap process for the development of new proangiogenic casein manganese nanoparticle or nano-formulations and their applications for the treatment of various diseases (such as wound healing and ischemic diseases especially limb ischemia) where angiogenesis plays an important role.

BACKGROUND AND PRIOR ART OF THE INVENTION:

Protein based metal nanoparticles are explored worldwide in different biomedical fields [ Verma, D., et al., Journal of pharmaceutics, 2018. 2018: p. 9285854-9285854]. Among several nanoparticles, manganese (Mn) based metal nanoparticles have attracted maximum attention for different biomedical applications due to various reasons [ Haque, S.,et al. Nanoscale, 2021. 13(39): p. 16405-16426],

Manganese is one of the essential micronutrient required for metabolism, growth, reproduction, etc. [ Li, L et al. Oxidative medicine and cellular longevity, 2018. 2018: p. 7580707-7580707]. Further, manganese is actively involved as a cofactor for many enzymes and one of the main components of manganese dependent superoxide dismutase [ Martinez-Finley, E.J., et al 2013, Springer New York: New York, NY. p. 1297-1303].

Several reports demonstrated the wound healing application of manganese through modulation of integrin [ Tenaud, I., et al., Br J Dermatol, 1999. 140(1): p. 26-34]. Manganese has also been reported to be involved in maintining mucopolysaccharide within the epiphyseal cartilage for the development of normal bones in chick [ Leach, R.M., et al, The Journal of Nutrition, 1962. 78(1): p. 51-56]. Further, manganese dependant MnSOD mediated by STAT3 have role in the stem cell renewal through maintaining the pluripotency of mouse embryonic stem cells [ Sheshadri, P., et al.,. Sci Rep, 2015. 5: p. 9516].

Several literature reports the different biomedical applications of manganese-based nanoparticles for drug delivery, antimicrobial, anticancer, antioxidants, nanozymes, photothermal therapy, biosensing, etc. [ Chen, Y., et al.,. Nanotechnology, 2020. 31(20): p. 202001; Lopez-Abarrategui, C., et al.,. International journal of nanomedicine, 2016. 11: p. 3849-3857; Gupta, R. et al. ACS Applied Nano Materials, 2020. 3(2): p. 2026-2037; Chowdhury, A., et al.. International Nano Letters, 2017. 7(2): p. 91-122; Hussain, S.M., et al.. Toxicological Sciences, 2006. 92(2): p. 456- 463; Qu, F., et al.. Taianta, 2017. 165: p. 136-142; Shaik, M.R., et al.. Saudi Journal of Biological Sciences, 2021. 28(2): p. 1196-1202; Jiang, D., et al.. Chemical Society reviews, 2019. 48(14): p. 3683-3704; Jankowski, J., et al.. Pios one, 2018. 13(7): p. e0201487 ].

However, therapeutic angiogenesis of manganese is not known in the prior art. Angiogenesis is the formation of new blood vessels from the pre-existing blood vessels and is one of the major processes during wound healing, limb ischemia, cardiovascular diseases, etc. [ Folkman, J.et al. Annu Rev Med, 2006. 57: p. 1-18]. There are various signaling molecules, growth factors (VEGF), cytokines that regulate the process of angiogenesisf Folkman, J.et al. Annu Rev Med, 2006. 57: p. 1-18]. Moreover, reactive oxygen species (ROS) is one of the mechanisms that drives angiogenesis [ Ushio-Fukai, M. et al. Cancer Letters, 2008. 266(1): p. 37-52]. It has been reported that VEGF- induced ROS enhances MnSOD within ECs that result in a feed-forward mechanism for triggering ROS by more H2O2 formation, thus increasing angiogenesis [ Ushio-Fukai, M. et al. Cancer Letters, 2008. 266(1): p. 37-52],

On the other hand, protein-based nanoparticles have been recently explored for their biocompatible nature, bioavailability, etc. There are some reports where bovine serum albumin has been used as biological template for the preparation of manganese nanoparticles. But the synthetic procedure involves critical conditions including long duration time, dialysis for 2 days for purification of the nanoparticles [ University, S., 2011, Sichuan University: China; China, National Center for Nanosccience and Technology China: 2021, China]. In the first report, the manganese nanoform is used for absorption, catalysis, bio-marking and other fields [ University, S., 2011, Sichuan University: China]. In the second report the manganese nanoparticle has been converted into a nano adjuvent using an antigen protein for enhancing the cellular internalization of the immune antigen and activate immune cells [ China, National Center for Nanosccience and Technology China: 2021, China].

On the other hand, casein is one such food degradable protein that is widely used as templating agent during the formation of nanoparticles [ Gl^b, T.K. et al, Topics in current chemistry (Cham), 2017. 375(4): p. 71-71]. Casein is the major milk protein that is economic, easily available, less toxic and stable making them to be referred to as GRAS (generally recognized as safe) protein [ Goulding, D.A., et al. 2020, Academic Press, p. 21-98]. The casein has the ability to bind to ions as well as to small molecules, have emulsification properties and water binding abilities [ Sadiq, U., et al. Foods (Basel, Switzerland), 2021. 10(8): p. 1965]. Casein nowadays is readily being used as encapsulation materials for different hydrophobic drugs and molecules because of its tendency to form micelles [ Sadiq, U., et al. Foods (Basel, Switzerland), 2021. 10(8): p. 1965]. The casein peptides are found to be flexible and randomly coiled forming intermolecular interactions due to the less frequency of the secondary structures. Henceforth, the casein is present in an energy favorable confirmation [ Glqb, T.K. et al, Topics in current chemistry (Cham), 2017. 375(4): p. 71-71]. Further, the decomposition temperature of casein is more than 200°C [ Lee, H., et al., Polymers, 2020. 12(9): p. 2078]. The casein-based nanoparticles will have potential therapeutic and diagnostic for various diseases [ Lohcharoenkal, W., et al., BioMed research international, 2014. 2014: p. 180549-180549].

As per reports, ischemic diseases such as limb ischemia are currently one of the most dreaded diseases globally leading to huge socio-economic losses. The conventional treatment therapies with different cytokines are usually employed [ Barui, A.K., et al., Biomed Mater, 2021. 16(4)]. Also, wound healing is one of the important applications of angiogenesis. Surgical wounds or traumatic injury in diabetes, immune deficiency, and so forth can be fatal because of the futility of conventional treatments where almost 40% of the cases lead to severe infection as well as mortality [ Rao, B.R., et al.,. ACS Applied Materials & Interfaces, 2021. 13(9): p. 10689-10704]. These conventional strategies have several limitations such as lesser bioavailability, non-specificity, high cost, etc. Therefore, as an alternative nanotechnology is being employed for the treatment of such diseases.

Therefore, in view of the above-mentioned limitations as well as drawbacks known in the development of nanoparticles/nanocomposites and considering the importance of angiogenesis for the treatment of wound healing and ischemic diseases, it is desirable to design an efficient, fast, clean, economically cheap biocompatible nanoparticles of manganese and method for the formation thereof. The nanoparticle is a casein-based manganese nanoparticles prepared by the interactions of manganese chloride (MnCh) and casein protein in the presence of sodium hydroxide (NaOH), as provided by the present disclosure. OBJECTIVES OF THE INVENTION:

The main objective of the present invention is to provide a casein manganese nanoparticle (abbreviated as CMNPs) using manganese chloride (MnCh) and casein protein in the presence of sodium hydroxide (NaOH) (pH around 8) overcoming the above-mentioned limitations known in the prior art.

The second objective is the characterization of CMNPs using several analytical tools (XRD: X- ray diffraction), DLS (Dynamic light scattering), FTIR (Fourier transform infrared spectroscopy), TGA (Thermogravimetric analysis), XPS (X-ray photoelectron spectroscopy), SEM (Scanning electron microscopy), TEM (Transmission electron microscopy), EDAX (Energy dispersive X- Ray analysis) and optimization.

In another objective of the invention described herein is to effectively illustrate the in vivo biocompatibility of casein manganese nanoparticle using hemolytic assay (after collection of red blood cells from C57BL/6J mice).

In vitro and in vivo therapeutic angiogenic activity of casein manganese nanoparticles is carried out using different cell lines (endothelial: HUVEC, EA.hy926; keratinocytes: HAEK cells), CAM assay and chick aorta assay, respectively.

Yet another objective of the present invention demonstrates the importance of CMNPs in vivo therapeutic angiogenesis in wound healing (in C57BL/6J mouse model using punch biopsy) and limb ischemia (BALB/c model). In summary, the objective of the present invention relates to the design and development of biocompatible casein manganese nanoparticles (CMNPs) for therapeutic angiogenic applications in wound healing and limb ischemia.

SUMMARY OF THE INVENTION:

The present invention provides a casein manganese nanoparticle and a simple, fast, efficient, economically cheap method for the preparation of casein manganese nanoparticles (CMNPs) that was formed by the combination of MnCh (as source of Mn) and casein protein (as biological template) in the presence of NaOH under stirring at room temperature.

In an aspect, the present invention provides a casein manganese nanoparticle (CMNPs) having size in the range of 50-100 nm. In an aspect, the present invention provides the casein manganese nanoparticles are rod-shaped structures in clustered form within the matrix of casein and having size of 50-80 nm.

In another aspect, the present invention provides the casein manganese nanoparticles are having a hydrodynamic diameter of ~ 300 to 324 nm, preferably ~ 312 nm and a zeta potential of ~ -22 mV.

In another aspect, the present invention provides the casein manganese nanoparticles are having FTIR stretching frequencies selected from group comprising upper spectra selected from 3417.15, 2117.19, 1613.46 and 612.15, middle spectra selected from 3750.33, 3449.18, 2923.90, 1634.76, 1372.15, 1163.26, 1057.87 and 668.88, and lower spectra selected from 3419.02, 2924.15, 2076.71, 1636.83, 1514.67, 1389.47, 1237.45, 1118.73, 1061.76, 950.06, 808.91 and 520.86.

In another aspect, the present invention provides the casein manganese nanoparticles are useful in therapeutic angiogenesis.

In another aspect, the present invention provides the casein manganese nanoparticles enhance endothelial cell proliferation through multi-regulatory pathway.

In an aspect, the present invention provides the method for preparing a casein manganese nanoparticle, the method comprising: a. preparing a solution A comprising 20 mM manganese chloride salt (MnCh) per 10 ml of water; b. preparing a solution B comprising 10 to 100 mg casein per 1 to 3 ml of water; c. adding the solution B into the solution A to obtain a reaction mixture; d. adding sodium hydroxide solution into reaction mixture obtained in step (c) with constant stirring to obtain casein manganese nanoparticles; and e. purifying the casein manganese nanoparticles obtained in step (d).

In another aspect, the present invention provides the method, wherein the addition of the sodium hydroxide solution is carried out dropwise with a constant stirring at a temperature in a range of 25°C to 35°C at a pH of 8 for 5 to 6 hours, and wherein the sodium hydroxide solution added is IM solution in an amount of 200-400pl.

In another aspect, the present invention provides the method, wherein the purification of casein manganese nanoparticles in step (e) is carried out by pelleting and repeated washing with water and by centrifuging at a speed of 15000-20000 rpm for 15 to 25 minutes at a temperature in a range of 10 °C to 20 °C.

In another aspect, the present invention provides the method, wherein the solution B consists of 66.5 mg casein per 1 to 3 ml of water.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1. illustrates (a) Cell cycle and (b) Annexin V-FITC analysis using Flow cytometry of HUVEC cells incubated with CMNPs at therapeutic dose of around 10 pg/ml. Cells treated with CMNPs showed increase in the S phase (DNA replication) of the cell cycle compared to the untreated indicating the proliferating nature of the nanoparticles. Apoptosis analysis showed no changes of the CMNPs treated cells with respect to untreated indicating their biocompatible nature; These experiments are performed thrice and represented as mean±SD. (c)Hemolytic assay is studied using mice blood indicated the hemocompatible nature of the CMNPs (5-100 pg/ml). The inset picture represents the % of hemolysis (with respect to red colour intensity) is more in positive (+ve) control in comparison to other treatments; whereas no hemolysis is observed in negative (- ve) control. These experiments are performed thrice and represented as mean±SD. (d) Chorioallantoic membrane assay: Column I: 0 h, Column II: 4 h, Row I: UT, Row II: VEGF, Row III: CMNPs 10 (CMNPs 10 pg/ml) treated embryo. (e)Determination of the effect of CMNPs on ex vivo endothelial tube formation through embryonic chick aortic arch assay. The aortic arch has been divided into UT, VEGF and CMNPs 10 (CMNPs 10 pg/ml). The aortic arch treated with CMNPs showed increased sprouting in comparison to untreated samples. VEGF is used as positive control for all the experiments.

Figure 2 illustrates the immunofluorescence study to check the (a) Ki-67 (b) PECAM-1/ CD31 (c) VEGFR2 (d) a- SMA expression in HUVEC cells, (a), (b), (c) and (d): Row I: UT, Row II: VEGF, Row III: CMNPs 10 (CMNPs 10 pg/ml); Column I: DAPI, Column II: (a) Ki-67 (b) PECAM-1/ CD31 (c) VEGFR2 (d) a- SMA; Column III: merged. All the pictures are taken using confocal microscope at 60X magnification. Scale bar= 10 pm. Higher expressions of Ki-67, PECAM-1/ CD31, VEGFR2 and a- SMA have been observed in the HUVEC cells treated with CMNPs as compared to the untreated ones. Figure 3 illustrates :(a-b) Determination of the intracellular ROS (H2O2 and O2 ) production in HUVEC cell line using DCFDA and DHE reagent, respectively, (a) and (b) Row I: DCFDA, Row II: DHE, Column I: UT, Column II: TBHP, Column III: CMNPs 10 (CMNPs 10 pg/ml), Column IV: CMNPs 50 (CMNPs 50 pg/ml), Column V: L-NAME, Column VI: L-NAME+CMNPs 10, Column VII: MnO2. Higher amount of hydrogen peroxide and superoxide anion production is observed upon treatment with CMNPs compared to untreated (UT) cells, the CMNPs treatment along with L-NAME treatment (eNOS inhibitor) have increased the production of the ROS species as compared to only L-NAME treatment indicating the role of eNOS in ROS production and vice versa. (c)Determination of nitric oxide production in HUVEC. Row III: Nitric oxide production, Column I: UT, Column II: VEGF, Column III: CMNPs 10 (CMNPs 10 pg/ml), Column IV: CMNPs 50 (CMNPs 50 pg/ml), Column V: L-NAME, Column VI: L-NAME+CMNPs 10, Column VII: Mn02. A higher amount of nitric oxide production is observed upon treatment with CMNPs compared to untreated (UT) cells. Moreover, the CMNPs treatment along with L-NAME treatment (eNOS inhibitor) have increased the production of the nitric oxide as compared to only L-NAME treatment indicating the role of eNOS in the pro-angiogenic activity of CM NPs. All the images are captured at 60 X magnifications with the help of confocal microscopy (Scale bar=10 pm).

Figure 4 illustrates: (a-b) Investigation of molecular mechanism of CMNPs through western blot analysis of VEGFR2, FGFR1, GLUL, MnSOD2, Phospho-eNOS at Ser 1171, eNOS, Phospho- Akt at Ser 473, Akt, Phospho-p38 MAPK at Thrl80/Tyrl82 and p38 MAPK in HUVEC incubated with UT, VEGF, L-methionine sulfoximine (MSO), CM NPs-5 (CMNPs 5 pg/ml), CM NPs- 10 (CMNPs 10 pg/ml), MSO+CM NPs-5 and MSO+CM NPs-10. P-actin is used as loading control, (c) Overall schematic representation of the entire mechanistic pathway for pro-angiogenic activity of CM NPs.

Figure 5 illustrates: (a) Representative images of in vivo wound healing study of CMNPs in C57BL/6J mice using punch biopsy model for day 0, 4 and 8. The groups are Row I: UT, Row II: Silverex, Row III: Vaseline, Row IV: 0.1% CM NPs, Row V: 1% CMNPs where number of animals in each group=4; (b) Representative LDIs of the wound area of mice captured at three different time points: Day 0, 4 and 10. The scale represents the flow of blood from low (blue) to high (red). The blue is also an indication of formation of thick layer skin over the wound which is relatively more in the CMNPs treated group compared to untreated; (c) histopathological analysis of isolated skin sections around the wound area stained with Hematoxylin and Eosin (H & E).

Figure 6 illustrates the immunofluorescence study of isolated skin sections of wound area of C57BL/6J mice using (a) ki-67 and (b) PECAM-1/CD31. The groups are Panel I and II: Column I: DAPI, Column III: Merged; Panel I: Column II: Ki-67; Panel II: Column II: PECAM-1/CD31; Row I: UT, Row II: Silverex, Row III: Vaseline, Row IV: 0.1% CM NPs, Row V: 1% CMNPs Increased expression (higher green fluorescence) of Ki-67 and PECAM-1/CD31 in response to CMNPs (0.1 and 1%) administration compared to the untreated group indicating CMNPs based induction of cell proliferation (Scale bar= 10 pm), (c) Western blot analysis of the isolated skin tissues from the wound area of VEGFR2, FGFR1, PDGFRa, NfKB and a-SMA. P-actin is used as loading control.

Figure 7 illustrates: (a) Representative LDI images of the BALB/c hind limbs subjected to ischemic surgery induction which are captured for day 0 (after surgery), 5, 10 and 15. The groups are; Row I: UT, Row II: Sham, Row III: MnCh, Row IV: CMNPs-5 (CMNPs 5mg/kg of b. wt. of mice), Row V: CM NPs- 10 (CMNPs lOmg/kg of b. wt. of mice). While dark blue color indicates low blood perfusion level, yellow to red color depicts high blood perfusion. The restoration of blood for the sham group is normal 1-week post- surgery. The CMNPs treatment (both the groups) led to faster amelioration of ischemia compared to control group; (b) Representative LDI images of recurring limb ischemic model induction the BALB/c mice captured for day 0 (after surgery), 5, 10, 15, 21 (before surgery), 21 (after surgery), 27 and 30; Western blot analysis of the (c) soleus and (d) gastrocnemius muscle for VEGFR2, FGFR1, NfKB and a-SMA. P-actin is used as loading control.

Figure 8 illustrates: (a-b) Experimental design for sighting study of CMNPs for toxicity in C57BL/6J mice; Acute toxicity study of both male (Row I) and female (Row II) C57BL/6J mice (c) weight variation analysis, feed intake and organ weight index; (d) Hematological parameters: routine CBC, platelets and routine WBC differential; (e) Biochemical parameters: heart, liver and kidney function parameters.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention also relates to the development of casein manganese nanoparticle formulation. The developed method is simple, fast, efficient, economically cheap process for the development of new proangiogenic casein manganese nanoparticle or nano-formulations and their applications for the treatment of various diseases (such as wound healing and casein manganese nanoparticle ischemic diseases especially limb ischemia) where angiogenesis plays an important role.

Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and “the” include plural referents unless the context dictates otherwise. Thus, for example, reference to "a nanoparticle" includes a plurality of such nanoparticles, and reference to "the step" includes reference to one or more steps and equivalents thereof known to those skilled in the art, and so forth.

The terminology and structure employed herein is for describing, teaching and illuminating some embodiments and their specific features and elements and does not limit, restrict or reduce the spirit and scope of the claims or their equivalents.

More specifically, any terms used herein such as but not limited to “includes”, “comprises”, “has”, “consists” and grammatical variants thereof is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The specification will be understood to also include embodiments which have the transitional phrase “consisting of’ or “consisting essentially of’ in place of the transitional phrase “comprising.” The transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim, except for impurities associated therewith. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure.

As used herein, the term “about” is used to indicate a degree of variation or tolerance in a numerical or quantitative value. It indicates that the disclosed value is not intended to be strictly limiting, and may vary by plus or minus 5%, without departing from the scope of the invention. Unless otherwise defined, all terms, and especially any technical and/or scientific terms, used herein may be taken to have the same meaning as commonly understood by one having an ordinary skill in the art.

Reference is made herein to some “embodiments.” It should be understood that an embodiment is an example of a possible implementation of any features and/or elements presented in the attached claims. Some embodiments have been described for the purpose of illuminating one or more of the potential ways in which the specific features and/or elements of the attached claims fulfil the requirements of uniqueness, utility and non-obviousness.

As used herein the terms “method” and “process” have been used interchangeably.

The following examples are provided to illustrate the present invention and should not be construed to limit the scope of the present invention.

Procurement details: Manganese chloride salt (MnCh), glycine, acrylamide, bis-acrylamide and sodium bicarbonate (NaHCCh) are purchased from HiMedia (India). Casein, sodium hydroxide (NaOH), MTT (3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide), trypsin, FBS: fetal bovine serum, DMEM: Dulbecco's modified Eagle's medium, penicillin, protease inhibitor cocktail (PIC), RIPA buffer, ribonuclease (RNase), PI: propidium iodide, DCFDA: 5-(and-6)- carboxy-2',7'-dichlorofluorescein diacetate, glycine, acrylamide, tris-base, ammonium persulfate, TBHP (tertiary butyl hydroperoxide), L-NAME (Nm-Nitro-L-argin ine methyl ester hydrochloride), wortmannin (Wort)and PBS: phosphate buffer saline are procured from Sigma Aldrich Chemicals, USA. Methanol (MeOH) is brought from Merck Specialties (India). Tween-20 is purchased from Amresco (USA). Triton X-100 and Bradford reagent are brought from Genetix Biotech Asia. Dimethyl sulphoxide (DMSO) is purchased from Rankem (India). [Methyl ³ H] -Thymidine was purchased from Perkin-Elmer Life and Analytical Sciences, USA. PVDF membrane is procured from Merck Milipore. All the experiments have been carried out in Milli-Q-grade water (18.2 MQ.cm).

Cell line: Primary cell line- Human umbilical vein endothelial cell (HUVEC) and its culture media (EBM) has been purchased from Lonza, USA. The other endothelial cell line EA.hy926 (human endothelial somatic hybrid cell lines) is received from our research collaborator, Dr. Suvro Chatterjee, Au-KBC, Chennai, India with the authorization of Steve Oglesbee, TCF, UNC Lineberger Comprehensive Cancer Center, NC, USA. Human adult epidermal keratinocytes (HAEK) are purchased from Himedia, India.

Antibodies: Primary antibodies anti-FGFRl, anti-PDGFR a, anti-VEGFR2, anti-aSMA, anti- CD31, anti-Ki67, anti-AKT, anti-phospho AKT, anti-MAPK, anti-phospho MAPK, anti- eNOS, anti-phospho eNOS, anti-MnSOD, anti-GEUE, anti-NfKB and anti-P-actin rabbit mAb antibodies as well as secondary goat anti-rabbit and anti-mouse IgG HRP antibodies are procured from Cell Signaling Technologies. Alexa Flour 488 anti-rabbit, FITC conjugated anti-mouse are purchased from Puregene. Protein ladder and chemiluminescent substrate are purchased from Thermo Fischer Scientific.

Detection Kits: Annexin V-FITC-labeled apoptosis detection kit is purchased from BD Biosciences, (San Jose, CA); respectively.

EXAMPLE-1: Method of preparation of the casein manganese nanoparticles (CMNPs): The synthesis of CMNPs involves the interaction between manganese chloride salt and casein in the presence of sodium hydroxide. Manganese chloride salt (MnCh: 20mM) is added to a beaker containing 10 ml water (solution A). Then in a separate beaker casein of different weight (10, 50, 50, 66.5, 100 mg) is added in water (1-3 ml) (solution B) for optimization and are designated as entry no # 1-5, respectively. Now, solution B is mixed with solution A followed by dropwise addition of sodium hydroxide solution (IM) (200-400pl) for 5-6 hours under constant stirring at room temperature (25 °C to 35 °C) at pH around 8. The appearance of dark brown color from white colored solution indicates the formation of CMNPs. After completion of the reaction, the resultant brown colored solution is pelleted and washed several times with MiliQ water by centrifugation (15000 to 20,000 rpm, 15 to 25, preferably 20 mins, 10°C to 20°C, preferably 15°C) using Sorvall- WX ultra 100 (Thermo scientific). The CMNPs are optimized through several reaction sets by keeping the MnCh constant (10 ml:20mM) and changing the volume of casein (10, 50, 50, 66.5, 100 mg; entry no #1-5, respectively) and sodium hydroxide (200-400 pl) with entry no. 4 being the optimized set. The appearance of dark coloured reaction mixture indicates the formation of optimized casein manganese nanoparticles. The alkaline pH around 8 due to sodium hydroxide helps in increase of the size of the casein micelle.

EXAMPLE-2: Characterization of casein manganese nanoparticles (CMNPs): X-ray Diffraction (XRD) Spectroscopy: The crystallographic structure and phase purity of the CMNPS is determined by X-ray powder diffraction method using Bruker AXS D8 Advance Powder X-ray diffractometer (where Cul<ak=l .5406 A radiation) [ Das, S., et al.,. Nanoscale, 2020. 12(14): p. 7604-7621]. The intense brown colored solution obtained after centrifugation is coated on a glass slide and submitted for XRD. The diffraction patterns are measured over a 20 range of 20° to 80°. The XRD pattern of CMNPs shows prominent diffraction peaks from 15° and 40° suggesting its crystalline nature. The peaks within 15°-40° can be distinctly indexed to (200), (400) and (211) diffraction planes. Similarly, the peaks within 40°-60° can be indexed to (411), (600) and (521) diffraction planes. The diffraction peaks for all reflections are consistent with the standard data files (JCPDS card PDF file no. 44-0141) of MnCh.Most of the peaks above 40° are not prominent may be due to the coating of casein on the outer surface of the manganese dioxide nanoparticles that hinders the penetration of the X-rays.

EXAMPLE-3: X-Ray photoelectron spectroscopy (XPS) analysis'. To determine the surface chemistry of the CM NPs, XPS analysis [KRATOS AXIS 165 with a dual anode (Mg and Al) apparatus using the Mg Ka anode] is done [ Das, S., et al.,. Nanoscale, 2020. 12(14): p. 7604- 7621]. XPS is used to determine the surface chemistry of the nanoparticles by analyzing the composition, electronic structure and oxidation states of the elements present in the material. The XPS survey spectrum of CMNPs indicates the binding energy (B.E) of C, O and Mn atoms. The B.Es. around 288.259, 530.995 eV correspond to the presence of Cis and Ols, respectively. Figurelc illustrates two spin orbit components, Mn 2pl/2 and Mn 2p3/2 which arise due to resolving the Mn 2p spectrum at B.E 653.188 eV and 641.748 eV respectively, validating the oxidation state of Mn as Mn02 in CM NPs. Additionally, the baseline of the curves touches the x- axis indicating the presence of a single oxidation state of Mn (in Mn02).

EXAMPLE-4: Scanning electron microscopy (SEM) analysis: The dark brown color powder obtained after centrifugation is used for SEM using SEM Hitachi S-3000N, Japan. The low- resolution SEM image of CMNPs display its overall surface morphology of randomly oriented nanorods. The SEM images of the CMNPs corroborate with earlier reports of MnC nanorods structures.

EXAMPLE-5: Transmission electron microscopy (TEM) analysis: The shape, size and morphology of the CMNPs are analyzed by the transmission electron microscopy (TEM: Tecnai G2 F30 S-Twin Microscope operated at 100 kV) [ Das, S., et al.,. Nanoscale, 2020. 12(14): p. 7604-7621]. The intense brown coloured powdered samples obtained after synthesis is diluted in 1:5 ratio with water, sonicated and a drop is placed onto a carbon coated copper grid. The grid is then allowed to dry and submitted for TEM analysis. The TEM analysis shows the shape, size and aggregation of CM NPs. The TEM images of CMNPS (higher magnification) and (lower magnification) indicate their rod-shaped structures in clustered form within the matrix of casein having sizes between 50-80 nm. The lattice fringes indicate the crystalline nature of MnCh in the CM NPs. The SAED pattern indicates their poly crystalline structure that are indexed to (200), (400), (411), (600) and (521) distinct planes. It is to be noted that the size of CMNPs measured by DLS (-300 nm) shows larger size than that of TEM (-80 nm) because DLS measures the entire hydrodynamic radius of particles in solution whereas TEM analyses the exact size of the solid particle. The rod-shaped CMNPs helps in faster internalization within the cells.

EXAMPLE 6: Energy dispersive atomic X-ray analysis: The EDAX analysis is performed to check the amount of manganese metal present within CM NPs. The EDAX detected that the rodshaped CMNPs contain maximum amount of Mn. The other constituents of casein (calcium, phosphorus), along with nitrogen, oxygen and carbon are also analyzed. The sample is mostly composed of manganese.

EXAMPLE-7: Dynamic light scattering (DLS) analysis: The DLS is used to measure the size and charge of CMNPs which forms one of the defining criteria for the formation of the nanoparticles. The hydrodynamic diameter and the zeta potential are measured Litesizer 500 particle analyzer (Anton Par)[ Das, S., et al.,. Nanoscale, 2020. 12(14): p. 7604-7621]. The sample to be measured is prepared by diluting 50pl of CMNPs in 950 ml of MilliQ water for both size and charge. The DLS analysis is a technique used to measure the hydrodynamic diameter and zeta potential of CM NPs. The hydrodynamic diameter of CMNPs is found to be ~ 300 to 324 nm with an average of ~ 312 nm, and the zeta potential is approximately -22 mV. The negative charge indicates less aggregation and more stability of the formed nanoparticles.

EXAMPLE-8: Fourier transformed infrared (FTIR) spectroscopy of nanocomposites: The fourier transformed infrared spectroscopy (FTIR: Thermo Nicolet Nexus 670 spectrometer) is performed to analyze the possible changes functional groups during the formation of the CM NPs [ Das, S., et al.,. Nanoscale, 2020. 12(14): p. 7604-7621]. The FTIR spectra is recorded in the diffuse reflectance mode having a resolution of 4 cm' ¹ in KBr pellets from range 400-4000 cm ¹ . The CMNPs obtained after synthesis and centrifugation are lyophilized and submitted for analysis. The FTIR analysis of MnCh, casein and CMNPs indicates the changes in the functional groups involved during the formation of CMNPs. The major IR stretching frequencies obtained from MnCh (upper spectra) are 3417.15, 2117.19, 1613.46 and 612.15, casein (middle spectra) is 3750.33, 3449.18, 2923.90, 1634.76, 1372.15, 1163.26, 1057.87 and 668.88 as well as CMNPs (lower spectra) are 3419.02, 2924.15, 2076.71, 1636.83, 1514.67, 1389.47, 1237.45, 1118.73, 1061.76, 950.06, 808.91 and 520.86. Some of the major IR peaks, observed in MnChand casein are stretched in case of CMNPs illustrating the involvement of active functional groups (alkanes, alkenes, amines, alkyl amines, aldehydes, ketones, alkyl halides, etc.) in the formation of CMNPs using sodium hydroxide of MnChusing casein as matrix.

EXAMPLE 9: Thermogravimetric and Differential Scanning Calorimetry:

The thermal stability along with the derivative thermogravimetric (DTG) analysis including the presence of water molecules in CMNPs are examined by TGA using TGA/SDTA851c, from METTLER TOLEDO, Switzerland at 10 °C/min from 25-800 °C under N2 atmosphere. The TGA pattern of CMNPs exhibits three weight losses within the temperature range 25 °C to 500°C. The first weight loss (-6.1%) around 100-200°C corresponds to loss of the absorbed moisture on the surface of CM NPs. The other two weight losses at around 230°C and 370°C correspond to -8.8% and 13.26%, respectively. The two weight losses indicate the presence of different types of binding of casein with CM NPs. Overall, CMNPs undergoes 20-30% weight loss upon thermal degradation from temperature range of 25°C to 500°C. The corresponding DTG curves indicate the exothermic decomposition of casein in CM NPs.

EXAMPLE-10: Cell viability assay: HUVECs are cultured in EBM complete media supplemented with 5% FBS and antibiotics. The experiments with HUVECs are performed in starved EBM media containing 0.2% FBS to synchronize the effect of treatments in all experiments. The in vitro cell viability assay indifferent cells (HUVEC, EA.hy926 and HAEK) is checked through MTT assay in accordance with the previous published literature [29] .The HUVEC cells are incubated with CMNPs (10-100 pg/ml), Casein (1-100 pg/ml) and MnCh (1- 100 pg/ml).The EA.hy926 incubated with CMNPs (10-100 pg/ml). The HAEK incubated with CMNPs (10-100 pg/ml), Casein (2-5 pg/ml) and MnCh (5-8 pg/ml) for 24 h. VEGF is used as positive control for all the experiments. The CMNPs does not show any cytotoxicity to the all the cells after 24 h incubation (in a dose dependent manner from 1-100 pg/ml) even at higher concentration indicating their biocompatible nature. Moreover, CMNPs at 10 pg/ml concentration improved the endothelial (HUVEC and EA.hy926) cell and keratinocyte (HAEK) growth as well as proliferation compared to untreated (UT). The results suggested the biocompatibility as well as the pro-angiogenic property of CM NPs.

EXAMPLE-11: [ ³ H]-Thymidine incorporation assay: The major indication for the proliferation of cells is the rapid amplification of DNA. Thymidine incorporation is one of the key processes occurring in the DNA formation since thymidine is among one of the nucleotide base pairs of DNA [ Nethi, S.K., et al.,. ACS Biomaterials Science & Engineering, 2017. 3(12): p. 3635-3645]. Briefly, HUVEC cells (5X10 ⁴ /well) are seeded in 24-well plate till they reach 80% confluency followed by serum starving for 6 hand treatment with CMNPs (lOpg/ml) for 24h to previous published literature. Later, the cells are washed with IX PBS twice and radioactive [ ³ H]- Thymidine (IpC) is provided for 4 h and incubated. The cells are then subjected to IX PBS wash (twice) followed by cell lysis with 0.1% SDS for 1 h. After that, 50pl of cell lysate and 50pl of scintillating oil are transferred to 96 well plates. The radioactivity of the prepared sample is measured in terms of counts per minute. The experiments are carried out in counts per minute in order to obtain a standard deviation. The results exhibited improved uptake of [ ³ H] -thymidine in HUVECs incubated for 24 h with CMNPs (5-50 pg/ml) as compared to untreated cells. VEGF is used as positive control. The inference of the results points to the increased DNA replication along with cell proliferation of HUVECs treated with CMNPs which corroborates with the cell viability assay.

EXAMPLE-12: Scratch wound assay: The scratch assay is done to check the proliferative effect of the CMNPs indifferent cells (HUVEC, EA.hy926 and HAEK) after scratch effect. Briefly, HUVEC cells (5X10 ⁴ /well) are seeded in 24-well plate till they reached 80% confluence [ Nethi, S.K., et al.,. Nanoscale, 2015. 7(21): p. 9760-70]. Briefly, (HUVEC, EA.hy926 and HAEK) cells (5X10 ⁴ /well) are seeded in 24-well plate till they reach 80% confluence. An in vitro wound model is generated using sterile micropipette tip by scratching on monolayer of cells. The remaining cells are washed with IX PBS to remove the floating cells and subjected to treatments; HUVEC cells treatment: i) untreated ii) CMNPs (lOpg/ml) iii) CMNPs (50pg/ml) iv) VEGF v) Mn02; EA.hy926: i) untreated ii) CMNPs (lOpg/ml) iii) VEGF ; HAEK i) untreated ii) CMNPs (lOpg/ml and 20(ig/ml) for 8 h. The bright field images of the scratched area of cells at time intervals of 0 and 8h are taken under an inverted microscope (Nikon, Tokyo, Japan) at 4X magnification. The wound closure is calculated with the help of IMAGE J software analysis (NIH, Bethesda, MD, USA).

The results clearly state that the CMNPs efficiently induce higher migration of the cells (HUVEC, EA.hy926 and HAEK) towards the scratch area in a time period of 8 h at 10 pg/ml, in comparison with the untreated control. CMNPs at different concentration (10 & -50 pg/mL) efficiently induce higher migration of cells towards the scratched area in 8 h as compared to untreated control. VEGF is used as positive control for all cells. Mn02 is used to compare between the commercial Mn02 and the synthesized CMNPs (with manganese in the Mn02 form).

EXAMPLE -13: Tube formation assay: The tube formation is one of the key characteristics of angiogenesis shown by the endothelial cells. The tube formation assay is performed in accordance with the previous published literature [ Nethi, S.K., et al.,. Nanoscale, 2015. 7(21): p. 9760-70]. Briefly, 15x103 HUVECs are seeded in a 48-well plate that is coated with matrigel. The cells are then treated with CMNPs (lOpg/ml and 50pg/ml) along with VEGF (10 ng/ml) as positive control. The cells gave rise to tube like extensions within 48 h. Following that, the images are captured using Olympus inverted microscope at 10 X magnification. The results exhibited that the CMNPs at a concentration of 1 Opg/ml helps in the elongation of the tubular like structure formation of the HUVECs in comparison with untreated, control indicating the pro angiogenic property of CM NPs. The VEGF is used as positive control.

EXAMPLE-14: Cell cycle analysis: Cell cycle analysis is performed using the flow cytometry technique. Briefly, the HUVEC are grown to confluence and incubated with CMNPs(l Opg/ml) for 24 h [ Nethi, S.K., et al.,. ACS Biomaterials Science & Engineering, 2017. 3(12): p. 3635-3645]. After that, the cells are trypsinized, fixed with 70% ethanol and stored at -20 °C for overnight. Next day, the cells are again washed with PBS, stained with propidium iodide (PI) solution supplemented with RNase and triton-X for 40 min under dark condition. Finally, the cells are washed with PBS and analyzed for cell cycle in a FACS Canto II, Becton Dickinson, San Jose, CA, U.S. Figure la depicts slight increase in S phase (DNA synthesis phase) cell population of the CMNPs treated HUVEC cells when compared with the untreated cells which might support the pro- angiogenic properties of the CM NPs.

EXAMPLE-15: Apoptosis study using FACS: Apoptosis assay is carried out in HUVEC cells using flow cytometry. Briefly, 1 x 10 ⁶ cells media are plated in 60 mm tissue culture dishes. The cells are treated with i) CMNPs (10 pg/ml) ii) VEGF (10 ng/ml) for 24 h and subjected to FACScan flow cytometry for apoptosis analysis using an Annexin V FITC kit, following the manufacturer's protocol after washing extensively with DPBS.

Figure lb shows no significant change in the CMNPs treated HUVEC cells in the apoptotic (early: Q4 and late: Q2) region compared to untreated cells, suggesting that CMNPs are non-apoptotic and biocompatible to endothelial cells.

EXAMPLE 16: Hemolysis assay: Briefly, 2ml of mice blood (C57BL6/J) is collected through retroorbital region. The erythrocytes are collected by centrifuging the sample at 3000 rpm, 10 min, 4°C using the Labocene centrifuge (model no:- Scanspeedo 1730R) [ Das, S., et al.,. Nanoscale, 2020. 12(14): p. 7604-7621]. The pellet is further washed thrice in DPBS buffer, and the supernatant is discarded. The stock solution is prepared by suspending the pellet in DPBS from where 0.1 ml of the erythrocyte sample suspension are added to 0.9 ml each of i) tap water (positive control) ii) DPBS buffer (negative controls) iii) CMNPs [solution made in DPBS as 10-100 pl from lOOmg/ml stock]. The samples are incubated in a water bath shaker for 90min at 37°C. Then the samples are centrifuged at 3000 rpm, 10 min at 4°C. The absorbance of the supernatant is measured at 514nm using the Synergy Hl multimode reader by collecting the supernatant.

% of hemolysis = (O.D. of NPs-O.D. of negative control)/(O.D. of positive control -O.D. of negative control) X100

The results in Figure 1c revealed that CMNPs (10-100 pg/ml) is hemocompatible even at higher doses as comparable to previous studies. The precursors of the CMNPs that is casein and MnCh is also biocompatible in nature.

EXAMPLE 17 '.Chorioallantoic membrane assay: The chorioallantoic membranes from the chick are widely used in various biological applications in order to check the in vivo angiogenesis, biocompatibility etc. [ Das, S., et al.,. Nanoscale, 2020. 12(14): p. 7604-7621]. Here, in this study, the fertile eggs are kept in an incubator at 37°C, 60-70 % humidity. On the fourth day of incubation, a small hole is created in the shell and a miniscule amount of the albumin is drained out using a micropipette. Then, a shell is broken and peeled from the top, a window to expose the embryo. The treatments of CMNPs are given, and the eggs are further incubated for 4 h in the incubator. The pictures are taken at 0 h and 4 h using Leica Stereo Microscope.The chorioallantoic membrane assay is performed to examine the effect of CMNPs on the blood vessel development. Figure Id exhibit angiogenic nature of CMNPs on blood vessels at 4 h after treatment as compared to untreated samples. VEGF is used as positive control.

EXAMPLE-18: Chick aorta assay: The aortic arch assay using chick aorta is performed as per published protocol [ Roy, A., et al.,. Mater Sci Eng C Mater Biol Appl, 2020. 115: p. 111108]. Fertile brown Vanraja chicken eggs are allowed for development in egg incubators under optimum conditions as mentioned above. On the 12th day of development, the eggs are opened to isolate the embryo and the heart is isolated under aseptic conditions. The aortic arches are dissected carefully from heart tissue and cut into small pieces or rings of equal size followed by washing thoroughly with DPBS. Further, these rings are placed in a 24 -well plate which is previously coated with extracellular matrix (Matrigel, Corning). It is then kept in an incubator to settle. Later treatments (Control, CM NPs: 1 Opg/ml and VEGF) ae mixed with DMEM and added to the wells. After 48 h of treatment time, images are captured to analyze the endothelial cell sprouting from the chick aortic arches using a Nikon Eclipse: TE -2000E (Japan) microscope bright field microscope.

Figure le exhibits that the CMNPs 10 pg/ml increased vascular sprouting compared to the untreated samples.

EXAMPLE-19: Immunocytochemistry analysis: The immunocytochemistry analysis is performed in the endothelial cells to determine the expression of pro-angiogenic proteins due to the effect of CMNPs [ Nethi, S.K., et al.,. Nanoscale, 2015. 7(21): p. 9760-70]. The HUVEC endothelial cells are seeded onto coverslips in a 6 well plate at around 50-60% confluence. The cells are serum starved prior to treatment and then incubated with CMNPs (10 pg/ml) and VEGF (10 ng/ml) for 24 h followed by processing for immunofluorescence. The cells are washed with IX PBS (three times), fixed with 4% paraformaldehyde for 10 mins, again washed with IX PBS, permeabilized with 0.2% Triton X followed by blocking the nonspecific sites using 3% bovine serum albumin in TBS-T buffer after PBS wash for 1 h at room temperature. Then the cells are incubated with anti-Ki-67, anti-CD31, anti-a-SMA, anti-VEGFR2 primary antibody overnight at 4 °C and the next day with goat anti-mouse IgG FITC conjugate as well as goat anti-rabbit Alexa Fluor 488 secondary antibody for 1 h in the dark at room temperature. The cells are washed several times with IX TBST to remove the unbound antibody solutions and incubated with DAPI for 10 min for nuclear staining. Finally, the fluorescence images of the stained cells are captured by using Nikon TiEclipse Confocal Microscope.

Figure 2(a-d) shows that incubation of HUVECs with the CMNPs shows more green fluorescence compared with the control untreated cells in case of all the markers (Ki67, CD31, a-SMA and VEGFR2) indicating their increased expression. CMNPs group has higher Ki-67 expression compared to the untreated cells, indicating enhanced cell proliferation. There is increased CD31/PECAM-1 expression after CMNPs treatment compared to untreated samples indicating their role in active angiogenesis. Enhanced VEGFR2 expression in the CMNPs group compared to the untreated control.

EXAMPLE-20: Determination of ROS generation: The detection of ROS is done as per our previous reports. The experiment is performed with HUVEC cells where they are seeded into a 96- well plate at a density of 5 x 10 ³ cells per well, cultured and serum starved before treatment. Next day, the cells are incubated with i) untreated ii) CMNPs (10 pg/ml) iii) TBHP (lOOpM) iv) L-NAME (200 pM) v) L-NAME + CMNPs (lOpg/ml) vi) Mn02 for both DCFDA and DHE analysis using Nikon TiEclipse Confocal Microscope [laser used, 404.2 nm, 488 nm].

For HUVEC cells the confocal microscopy results showed that cells treated with CMNPs exhibited more ROS generation (H2O2, O2 ~) compared to untreated samples as in Figure 3(a-b). Further, the HUVEC cells when treated with L-NAME (eNOS inhibitor) together with CMNPs produced more ROS than only L-NAME treated samples. Therefore, the results overall suggest the role of CMNPs in generating higher eNOS for production of more intracellular ROS (positive feedback loop) indicating their important roles during angiogenesis.

EXAMPLE-21: Nitric oxide production DAF-2DA, a non-fluorescent dye having good cell permeability, can measure the NO levels within living cells. The diacetate moieties present in it get hydrolyzed by the intracellular esterases which on reacting with NO get converted to a fluorescent triazole derivative whose fluorescence can be measured at an excitation at 488 nm with emission at 515 nm. The assay is performed to check the NO generation capacity of the CMNPs within the endothelial cells [ Nethi, S.K., et al.,. Nanoscale, 2015. 7(21): p. 9760-70]. The HUVEC cells are seeded in a 96 well plate at about 40-50% confluence. The cells are serum starved before incubation with i) untreated ii) CMNPs (10 pg/ml) iii) TBHP (100 pM) iv) L-NAME (200 pM) v) L-NAME + CMNPs (lOpg/ml) vi) Mn02. The NO detection is performed as per the manufacturer’s protocol (Sigma Aldrich). Later the fluorescence is measured using Nikon TiEclipse Confocal Microscope for DAF fluorescence imaging.

Figure 3c represent the fluorescence images of HUVEC cells incubated with the following treatments: (a) control, (b) VEGF, (c) CMNPs 10 (d) L-NAME, (e) L-NAME +CM NPs, (g) MnO ₂ for 24 h followed by incubation with DAF-2DA as per the manufacturer’s protocol. The images clearly illustrate that green fluorescence is increased when the cells are treated with CMNPs as compared to the untreated control experiments. The feeble green fluorescence of untreated HUVEC cells appears may be due to the endogenous NO. The positive control treatment VEGF shows some amount of green fluorescence that supports the NO formation. In order to confirm the role of eNOS in NO production, the endothelial cells are incubated with an eNOS inhibitor L- NAME in the absence and presence of CM NPs. The results show little green fluorescence in cells due to the presence of L-NAME. However, the green fluorescence improved after the addition of CM NPs.

EXAMPLE-22: Western blot analysis'. The results in Figure 4 (a-b) show an increased level of p-38 MAPK, eNOS phosphorylation in HUVEC cells treated with 10 pg/ml CMNPs compared to untreated samples suggesting the pro-angiogenic nature of CM NPs. There is increase in phosphorylation of Akt when HUVEC is treated with CMNPs at 5 pg/ml concentration. Overall, these results suggest an Akt-eNOS-MAPK signaling involved behind the angiogenic activity of CM NPs. Further, the other pro-angiogenic factors like VEGFR2 and FGFR1 revealed an expression in the HUVEC cells when treated with CMNPs compared to the untreated cells. Since the nanoparticle is manganese containing, therefore, its role in MnSOD dependent angiogenesis has been explored. The expression of MnSOD is increasing in the HUVEC cells upon treatment with CMNPs (10 pg/ml). This data also corroborates with the insilico analysis data where manganese directly interacts with the MnSOD and the co-localization data where CMNPs are found to localize in the mitochondria more where endothelial MnSOD is located. Also, the insilico data reveals interaction of manganese with glutamine synthetase. Glutamine synthetase is involved in the synthesis of amino acid glutamine which helps in the endothelial cell mobility. Here, to inhibit the activity of glutamine synthetase L-methionine sulfoximine (MSO) inhibitor is used which does not decrease the concentration of glutamine synthetase but its activity might have decreased. The CMNPs have increased the concentration of glutamine synthetase, which may have resulted in increased mobility of endothelial cells as analyzed by scratch assay as well as enhanced expression of phosphorylated p-38 MAPK.Therefore, the scheme in Figure 4c represents the overall signalling pathway of the pro- angiogenic CM NPs.

EXAMPLE-23: In vivo wound healing study: The wound healing study in mice is performed based on approval of the Institutional Animal Ethics Committee (IAEC/33/2018), CSIR-IICT, Hyderabad, India [ Nethi, S.K., et al.,. ACS Biomaterials Science & Engineering, 2017. 3(12): p. 3635-3645]. C57BL6/J mice are employed for establishing an in vivo wound healing model. Briefly, 20-25gC57BL/6J female mice are used for the experiments where each group is n=3 (Group I: control; Group II: silverex (positive control); Group III: vaseline (vehicle control); Group IV: 0.1 % CMNPs Group V: 1 % CM NPs). The hair on the dorsal surface of each mouse is removed using commercially available hair remover cream (Veet). Further, a circular wound is created on the exposed surface of skin using a 6 mm punch biopsy by removing the skin. CM NPs- 0.1% and 1% treatment is preformulated using commercially available Vaseline cream. CMNPs are weighed, mixed in Vaseline base cream (1% w/w) for a fine paste and applied around the mouse wound (made by punch biopsy). All the treatments are applied around the wound area in alternate days (day 1, 3, 5 and 7) after creating the wound.

The results in Figure 5a indicate enhanced closure of the wound in the mice treated with CMNPs compared to vehicle control.

EXAMPLE-24: Laser doppler of wound area: To analyze the wound area recovery, the blood perfusion rate of wound area is measured using MOOR LDI2-HR laser Doppler imager (Moor Instruments, UK) [ Barui, A.K., et al., Biomed Mater, 2021. 16(4)]. The laser Doppler images for all the samples are captured at four time points i) before wound at day 0, ii) after wound at day 0 (by punch biopsy), iii) day 4, iv) day 10. The blood perfusion (mean flux value) is then analyzed at the region of interest (ROI) near the specified wound area of all the treatment mice groups using MOOR LDI2-HR laser Doppler imager software. Figure 5b shows that on day 0 the blood perfusion increased after creation of wound (redder in colour, usually presence of skin results in blue appearance in the blood perfusion rate) in all the groups, which gradually normalized through day 4 and finally on day 10 to blue in colour. Therefore, indicating the formation of skin layers over the wound. The decrease in the blood perfusion rate is faster in the CMNPs treated group compared to the untreated indicating its active role in healing as well as skin formation.

EXAMPLE 25: Skin histopathological analysis: After the observation period, the mice are sacrificed, dissected the wound area and collected in 4%paraformaldehyde solution [ Nethi, S.K., et al.,. ACS Biomaterials Science & Engineering, 2017. 3(12): p. 3635-3645]. This skin tissue is embedded in paraffin wax blocks from where thin sections (3 pm thicknesses) of the tissue are cut, mounted on to clean microscopic glass slides, washed and stained with hematoxylin and eosin (H&E) as per our previous reports. The sections are randomly analyzed for angiogenesis measurement by a certified pathologist, through observing the number of vessels at selected wound area section under HPF (40X).

The histological analysis reveals the anatomical changes of the skin sections in the groups of mice through H &E stained transverse skin sections as seen in Figure 5c. CMNPs (0.1 and 1%) treated groups of mice demonstrated normal sebaceous glands, hair follicles, mild thickening hyper keratosis of epidermis, dermal layer of skin as well as moderate vacuolar degeneration in epidermal layer. The untreated group on the other hand shows mild vacuolar, granulocytic and degenerative changes in epidermal layer. The Silverex (positive control) also depicts normal epidermal as well as dermal layer of skin, sebaceous glands and hair follicles. The vaseline (vehicle control) shows moderate vascular degeneration in the epidermis but degenerative changes are observed in the sebaceous glands.

EXAMPLE 26: Immunofluorescence of wound skin'. After sacrificing the mice, the wound skin tissues of all the groups are collected for immunohistochemistry analysis [ Barui, A.K., et al., Biomed Mater, 2021. 16(4)]. The muscle tissue samples are washed in DPBS followed by fixation with 4% PFA at 4°C for 48 h. Eater, the tissues are again washed with DPBS and stored at 4°C in 20% glycerol. Again, the samples are embedded in paraffin, sectioned (5 pm thickness) which is mounted on microscopic slides. The slides are dipped in xylene for 5 mins (3 times), then decreasing concentration of ethanol for 5 mins each (100, 90 and 70 %), then warmed in 10 mM sodium citrate buffer (pH=6) for 10 mins and allowed to cool completely. The slides are washed with MiliQ, blocked with 5% bovine serum albumin in TBS-T buffer for 1 h at room temperature. The tissue sections are then incubated with primary antibodies (anti-Ki-67 and anti-PECAM- 1/CD31) for overnight at 4 °C and the next day with goat anti -rabbit Alexa Fluor 488 secondary antibody for 1 h in the dark at room temperature. The cells are washed several times with IX TBST to remove the unbound antibody solutions and incubated with DAPI (4’,6-diamidino-2- phenylindole) for 10 min for nuclear staining. Finally, the fluorescence images of the stained cells are captured by using Nikon TiEclipse Confocal Microscope.

Figure 6a depicts higher expression levels of Ki-67 nuclear marker in the CMNPs skin treatment groups compared to others, indicating that CMNPs augment cell proliferation resulting in the formation of new capillaries and eventually skin. Again, Figure 6b illustrates higher levels of PECAM-1/CD31 expression in the CMNPs treatment groups as compared to untreated.

EXAMPLE 27: Western blot of skin tissues: The western blot analysis was executed following our previously reported article [ Nethi, S.K., et al.,. ACS Biomaterials Science & Engineering, 2017. 3(12): p. 3635-3645]. Wound tissue samples for all the mice treatment groups are used for western blot analysis for protein estimation in order to determine the signaling pathway. The total protein is isolated from the skin tissue samples. The protein samples are prepared in RIPA buffer (Radio immunoprecipitation assay buffer) containing protease inhibitor cocktail (PIC) in 1000: 10 ratio. The samples are centrifuged at 12,000 rpm/ 4°C/ 20 min and the clear supernatant is collected. The total concentration of proteins in the supernatant is calculated by Bradford assay. Then 50 pg protein of all samples (equal concentration) are separated on a 10% SDS-PAGE which is then transferred to a poly(vinylidene difluoride) [PVDF] membrane (Merck Millipore). Nonspecific sites on the proteins are blocked using 3% BSA for 1 h 30 min and washed thrice with IX TBST (tris buffered saline with Tween 20). The membrane is then incubated with the respective primary antibodies; Wound healing: anti-VEGFR2, anti-FGFRl, anti-PDGF, anti-NFkB, anti-a SMA. The anti-P-actin is used as protein loading control for all the experiments. The dilutions are done in accordance with the manufacturer’s instructions for overnight at 4°C. The membrane is further washed with IX TBST solution three times followed by incubation with goat anti- rabbit/mouse IgG HRP antibodies at room temperature for 1 h. The immunoblot is developed by chemiluminescence instrument (Fusion solo S, Vilber Lourmat) using Super signal west pico plus chemiluminescent substrate (Thermo Scientific). The western blot analysis in Figure 6c of the isolated skin tissues including all the treatment groups illustrate the probable mechanistic pathways involved in the healing of wounds by CM NPS. Since the CMNPs are pro-angiogenic in nature, it helps in healing the wound through angiogenic pathway. The CMNPs reveals the increase in expression of proteins such as VEGFR2, PDGFRa, FGFR1, NFkB and a-SMA with respect to untreated samples.

EXAMPLE 28: Hind limb ischemia in mice model'. Unilateral hindlimb ischemia model is established in BALB/c mice through femoral artery ligation [ Barui, A.K., et al., Biomed Mater, 2021. 16(4).]. To develop the hind limb ischemia model, the animals are first anesthetized with intraperitoneal (i.p.) administration of ketamine (100 mg/kg b.w.)/xylazine (10 mg/kg b.w.) cocktail solution. A small incision of ~2 cm is made from knee toward the medial thigh, followed by tracing of femoral artery. The artery is then separated from femoral nerve and vein, after removing the subcutaneous fat. The femoral artery is ligated by at both proximal (near to groin) and distal end (close to knee), followed by closure of incision using suture. To investigate the therapeutic potential of CMNPs in hind limb ischemia, the animals are divided into five groups (n = 3-4): i) Sham (non-ischemic: only femoral artery exposure without ligation, no treatment), ii) Control (ischemic, no treatment) iii) MnCh (ischemic, MnCh: 10 mg/kg b.w.), iv) CM NPs-5 (ischemic, CM NPs: 5 mg/kg b.w.), and v) CM NPs-10 (ischemic, CM NPs: 10 mg/kg b.w.). CMNPs are intraperitoneally injected into mice at different time points (day-1, day- 3, day- 5 and day-7), post-ischemia (day-0). After four consecutive administrations of CMNPs on alternate days for 1 week, no further dose is given for another recovery week and the animals are sacrificed on day- 15, followed by collecting tissue samples for further experiments. To examine the CMNPs induced recovery from ischemic condition, blood perfusion analysis is carried out at the hind limb of mice using moorLDI2-HR Laser Doppler Imager (moor Instruments, UK). For this study, laser Doppler images (LDI) of all the groups are captured at four different time points (day 0, 5, 0 and 15). The blood perfusion (mean flux value) is then analyzed at the region of interest (ROI) close to the footpad of hind limbs of ischemic (control, MnCh, CM NPs-5 and lOmg/kg) and nonischemic (Sham) mice, employing moorLDI2-HR Laser Doppler Imager software. Similarly, recurring limb ischemic model is created having only untreated and CM NPs-10 as samples. The study is carried out in a similar manner to the 15 days surgery. Then on 21 ^st of the experiment, second ischemic injury is done on the same limb and carried up to 30 ^th day for the termination of the experiment. The Laser doppler results in Figure 7a indicate enhanced recovery of the hind limb ischemia in the mice treated with CMNPs compared to other groups. Even the recurring experiment in Figure 7b showed increase in the blood perfusion in the recurring CM NPs-10 group compared to recurring untreated.

EXAMPLE 29: Western blot of skin tissues: The western blot analysis was executed following our previously reported article [ Nethi, S.K., et al.,. ACS Biomaterials Science & Engineering, 2017. 3(12): p. 3635-3645]. For the limb ischemic study, the muscles tissues gastrocnemius and soleus are used for western blot analysis to explore the underlying mechanistic pathway for the recovery of limb ischemia. The total protein is isolated from the skin tissue samples. The protein samples are prepared in RIPA buffer (Radio immunoprecipitation assay buffer) containing protease inhibitor cocktail (PIC) in 1000: 10 ratio. The samples are centrifuged at 12,000 rpm/ 4°C/ 20 min and the clear supernatant is collected. The total concentration of proteins in the supernatant is calculated by Bradford assay. Then 50 pg protein of all samples (equal concentration) are separated on a 10% SDS-PAGE which is then transferred to a poly (vinylidene difluoride) [PVDF] membrane (Merck Millipore). Nonspecific sites on the proteins are blocked using 3% BSA for 1 h 30 min and washed thrice with IX TBST (tris buffered saline with Tween 20). The membrane is then incubated with the respective primary antibodies; limb ischemia: anti-VEGFR2, anti- FGFR1, anti-NFkB, anti-a SMA. The anti-P-actin is used as protein loading control for all the experiments. The dilutions are done in accordance with the manufacturer’s instructions for overnight at 4°C. The membrane is further washed with IX TBST solution three times followed by incubation with goat anti-rabbit/mouse IgG HRP antibodies at room temperature for 1 h. The immunoblot is developed by chemiluminescence instrument (Fusion solo S, VilberEourmat) using Super signal west pico plus chemiluminescent substrate (Thermo Scientific).

The western blot analysis in Figure 7(c-d) of the limb ischemic muscles (soleus and gastrocnemius) including all the treatment groups illustrate the probable mechanistic pathways involved in the recovery of limb ischemia by CM NPs. Since the CMNPs are pro- angiogenic in nature, it helps in recovery of limb ischemia through angiogenic pathway. The CMNPs reveals the increase in expression of proteins such as VEGFR2, FGFR1, NFkB and a-SMA (only in gastrocnemius) with respect to untreated samples. EXAMPLE 30: Sighting study for toxicity. Sighting study is carried out in C57BL/6J mice to determine the maximum tolerated dose (MTD) for CMNPs [ Nethi, S.K., et al.. Advanced Therapeutics, 2021. 4(6): p. 2100016.]. From Figure 8a it is observed that all the mice survived up to 2 weeks when injected with CMNPs in the dose range of 5-300 mg/kg b.w. Further, administration of 2000 mg/kg b.w. dose of CMNPs to the mice also showed good survivability with no evident pathologies up to 2 weeks. Hence, the above observations suggest that the MTD of CMNPs in C57BL/6J mouse by i.p. administration is > 2000 mg/kg b.w.

EXAMPLE 31: Acute toxicity study'. Acute toxicity study is proceeded for two weeks in both sexes of mice (treatment: n=6 for each male and female; control: n=3 for each male and female) using MTD of 2000 mg/kg b.w. dose of CMNPs in a single i.p. administration. Further, the analysis of body weight variation, feed intake, organ index, hematology and serum biochemical analysis are carried out as per the standard protocol.

The Table in Figure 8b represents the overall workflow for the acute toxicity study. Figure 8c shows the body weight variation, feed intake and organ index histogram where no changes are observed in the CMNPs treated group as compared to control. The hematological parameters (routine CBC, platelets and routine WBC differential) in Figure 8d shows no changes in the CMNPs treated group as compared to control. Further, the biochemical parameters in Figure 8e shows not much changes in the CMNPs treated female group as compared to control. The CMNPs male treated group shows some increase in triglycerides and aspartate levels as compared to untreated.

ADVANTAGES OF THE PRESENT INVENTION:

1. In the present invention, Inventors have prepared a new casein manganese nanoparticle (CMNPs) using casein as a template for formation of nanoparticles.

2. The present invention is simple, fast, clean, efficient, and economically cheap.

3. The CMNPs are stable in physiological environments and biocompatible towards in vivo systems.

4. The CMNPs demonstrate an escalated angiogenic activity in vitro towards different endothelial as well as in vivo through CAM and chick aorta assay. 5. The angiogenic CMNPs show wound healing property through in vitro assays towards keratinocyte cells and in vivo experiments in mouse model.

6. The angiogenic CMNPs show recovery of hindlimb ischemia in mice model.

7. Detailed studies shows that the CMNPs enhances endothelial cell proliferation through multi regulatory pathway mainly by ROS generation and propagating nitric oxide mediated pathway.

8. The present invention presents a simple approach to prepare casein-based biocompatible manganese nanoparticles having multiple applications and great potential in angiogenesis for future times.