Nanoparticle compositions

Abstract

The present invention relates to natural nanocarriers or nanoparticles with biocompatible, biodegradable, hypoallergenic, environmentally and physiologically acceptable properties. More in particular, it can be stated that the present invention relates to natural biopolymer nanocarriers, nanoparticles or nano-spheres with biocompatible, biodegradable, environmentally and physiologically acceptable properties as vehicles for physiological and environmentally safe delivery of active ingredients. The present invention also relates to nanoparticle compositions, methods of making the nanoparticles and nanoparticles compositions, as well as uses thereof.

Description

[0001] Attorney Reference No: 6666-P008-SAR-PCT

[0002] TITLE: NANOPARTICLE COMPOSITIONS

[0003] INTRODUCTION

[0004] [1] A multidisciplinary industry such as nanotechnology, frequently relies on nanostructures and nano-formulations made out of polymers such as natural biopolymers for different applications. Such nanostructures and nano-formulations represent functional materials with wide-spectrum of uses in human, agrochemical, veterinary, pharmaceuticals, food industry, water purification and others. Challenges presented by new products and reformulating new materials and chemicals aimed at solving global problems such as environmental restoration, energy reduction, zero-waste technologies, health care and improvement of lifestyle are continuously faced by the nanotechnology industry. Naturally derived polymeric materials (such as collagen, cellulose, starch, chitin, chitosan, alginate, gelatin, lignin) as precursors for the design and production of nanostructures and nano-formulations, as nanocarriers, have gained popularity as a consequence of their tunable physicochemical properties, unique morphology, particle dimension and enhanced bifunctionalities such as biocompatibility, biodegradable and bioactive carriers. Such nanocarriers, can be used for food packaging materials, energy storage, cosmetics, thermal / light stabilizers, drug delivery matrices, UV absorbers, hybrid nanocomposites, antioxidant and antibacterial agents.

[0005] [2] Lignin-based nanoparticles provide high added value opportunities. Lignin-based hydrogels and nano-formulations have attracted increased interest due to their applicability as UV blockers, antibacterial agents and drug delivery systems benefiting from their biocompatibility, biodegradability, low cytotoxicity, satisfactory antioxidant potential and sufficiently reactive functional groups allowing chemical modifications.

[0006] [3] However, biopolymer nano-formulations, for example made of cellulose and chitin, are characterised with poor mechanical strength, unstable structure, especially after implantation / transplantation, hydrophobicity, reduced moisture resistance and lack of solubility, rendering them potentially less desirable biopolymers.

[0007] [4] Also, although nano-formulations made from new natural, biocompatible, biodegradable and non-toxic ingredients display greater homogeneity and stability, during manufacture, these frequently require the presence of undesirable chemicals such as acids and / or bases, resulting in impeded quality of suspensions with reference to for example poor viscosity, stability, ease of encapsulation of an active ingredient and unpredictable release of encapsulated functional compounds.

[0008] [5] Reliance on toxic chemicals (such as tetrahydrofuran) further limits the applicability of known nano-formulations in for example biomedicine, pharmaceutical industry and food technology due to the manifestation of unpredictable side effects (Ali et al., 2020; Worku et al., 2023 and Mishra et al., 2019). Attorney Reference No: 6666-P008-SAR-PCT

[0009] Another problem is the inclusion of complex, indirect chemical reactions and processes that require expensive equipment (Qian et al., 2014; Tardy et al., 2018 and Tse et al., 2022).

[0010] [6] There is thus a need for simpler, safer and improved, for instance, in terms of physicochemical characteristics nano-formulations, characterised with better dispersibility, enhanced adhesive properties, improved encapsulation efficiency, sustained and predictable release of the encapsulated active compounds and enhanced biological activities such as biocompatibility, biodegradability, satisfactory antimicrobial and antioxidant properties.

[0011] [7] There is particularly a need for methods and processes not requiring addition of chemical compounds which would cause the presence of undesirable chemical substances in the end product.

[0012] 18] The invention therefore relies on the experiments described herein and offers new means and tools for addressing at least some of the above described shortcomings in the field of active ingredient nanoparticle encapsulation with physiological and environmentally safe delivery.

[0013] SUMMARY OF THE INVENTION

[0014] [9] In general terms, it can be stated that the present invention relates to the field of nanostructures, nano-formulations, nanocarriers, nanoparticles or nano-spheres. In particular, it can be stated that the present invention relates to natural nanostructures, nano-formulations, nanocarriers or nanoparticles. More in particular, it can be stated that the present invention relates to natural nanostructures, nano-formulations, nanocarriers, nanoparticles or nano-spheres with biocompatible, biodegradable, hypoallergenic, environmentally and physiologically acceptable properties. Even more in particular, it can be stated that the present invention relates to natural, nanostructures, nano-formulations, nanocarriers, nanoparticles or nanospheres with biocompatible, biodegradable, environmentally and physiologically acceptable properties as vehicles or conduits for physiological and environmentally safe delivery of active ingredients (Al). The present invention also relates to natural nanostructures, nano-formulations, nanocarriers, nanoparticles or nano-spheres comprising compositions or suspensions, methods of making the compositions or suspensions, as well as uses thereof.

[0015]

[0010] According to an aspect of the present invention, there is provided a nanoparticle composition for delivery of an active ingredient (Al), the composition comprises an organic acid, biopolymer and at least one Al.

[0016]

[0011] In some embodiments of the present invention, there is provided a nanoparticle composition comprising an organic acid, wherein the organic acid comprises one or more selected from the group consisting of lactic acid, citric acid, acetic acid, linoleic acid, gluconic acid, glucuronic acid, formic acid, and butanoic acid. Attorney Reference No: 6666-P008-SAR-PCT

[0017]

[0012] In some embodiments of the present invention, there is provided a nanoparticle composition wherein the biopolymer comprises one or more selected from the group consisting of lignin, chitin, chitosan, cellulose, starch, hemicellulose, amylose, actin, fibrin, collagen, fibroin and keratin.

[0018]

[0013] In some embodiments of the present invention, there is provided a nanoparticle composition wherein the biopolymer is a co-polymer of lignin and chitin.

[0019]

[0014] In some embodiments of the present invention, there is provided a nanoparticle composition, wherein the at least one active ingredient is one or more selected from the group consisting of a pharmaceutical product or compound, an agrochemical product or compound, a phytochemical product or compound, a cosmetic substance, a food constituent or a dietary supplement.

[0020] 115] In some embodiments of the present invention, there is provided a nanoparticle composition, wherein the organic acid, the biopolymer and the at least one active ingredient of the nanoparticle composition have been mechanically processed into a stable aqueous nanoparticle suspension.

[0021]

[0016] In some embodiments of the present invention, there is provided a nanoparticle composition wherein the organic acid, the biopolymers and the at least one active ingredient are biodegradable.

[0022]

[0017] According to an aspect of the present invention, there is provided a method for producing a biodegradable lignin-chitosan co-polymer nanoparticles composition, the method comprising the steps of: a. supplying an aqueous solution comprising lignin; b. contacting the solution of step a. with an antisolvent solution to form a pre-mixture; c. to the pre-mixture adding organic acid dissolved chitosan to form a mixture; and d. treating the mixture to produce lignin-chitosan nanoparticles suspension, wherein the antisolvent solution comprises at least one active ingredient.

[0023] [18| In some embodiments of the present invention, there is provided a method for producing a biodegradable lignin-chitosan co-polymer nanoparticles composition, wherein the step of treating the mixture comprises mechanical processing into a stable aqueous nanoparticle suspension.

[0024]

[0019] In some embodiments of the present invention, there is provided a method for producing a biodegradable lignin-chitosan co-polymer nanoparticles composition, wherein the aqueous nanoparticle suspension comprises lignin-chitosan nanoparticles, and wherein the lignin-chitosan co-polymer nanoparticles contain the active ingredient. Attorney Reference No: 6666-P008-SAR-PCT

[0025]

[0020] According to an aspect of the present invention, there is provided a biodegradable lignin- chitosan co-polymer nanoparticles composition comprising nanoparticles with a diameter in the range of about 10 nm to about 300 nm.

[0026]

[0001] According to an aspect of the present invention, there is provided a method for delivery of at least one active ingredient, the method comprising the steps of: a. providing a nanoparticle composition as described herein; b. formulating the nanoparticle composition; and c. administering the formulated composition of step b..

[0027]

[0022] In some embodiments of the present invention, there is provided a method for delivery of at least one active ingredient, wherein the formulated nanoparticle composition comprises at least one acceptable additive, palatant, excipient, binder, diluent, disintegrant, and / or adjuvant.

[0028]

[0023] In some embodiments of the present invention, there is provided a method for delivery of at least one active ingredient, wherein the nanoparticle composition is formulated for administering to a subject using one or more of the group of routes of administration consisting of per oral, intravenous, parenteral, rectal, nasal, topical (e.g., transdermal and intraocular), intravesical, enteral, vaginal, transurethral, intradermal, aural, intralesional, endoscopical, transmucosal, sublingual and intestinal.

[0029]

[0024] In some embodiments of the present invention, there is provided a method for delivery of at least one active ingredient, wherein the nanoparticle composition is formulated for agricultural administration using one or more of the group of routes of administration consisting of hydraulic spraying, backpack spraying, basal trunk spraying, aerial spraying, injection, soil injection, soil incorporation, ropewick and wiper treatment.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031]

[0025] FIG. 1. Transmission electron microscope (TEM) analysis of lignin-chitosan co-polymer nanoparticles.

[0032]

[0026] FIG. 2. FTIR spectra of the pure biopolymers, lignin and chitosan, and nanoparticles.

[0033]

[0027] FIG. 3. Concentration of surface acid functional groups is determined by potentiometric titration.

[0034]

[0028] FIG. 4. Antioxidant activity - DPPH and ABTS spectrophotometric assays are applied for determination of the scavenging activity of unloaded and loaded nanoparticles, pure chitosan and the bioflavonoid morin. Attorney Reference No: 6666-P008-SAR-PCT

[0035]

[0029] FIG. 5. In vitro release kinetic curves of morin from morin-encapsulated lignin-chitosan copolymer nanoparticles in both simulated physiological compartments.

[0036]

[0030] Figure 6. Methodical and experimental scheme representing a protocol for the synthesis and characterization of lignin / chitosan nanoparticles.

[0037]

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0038]

[0032] Throughout this disclosure, various scientific publications, patents and published patent application or granted patents are referenced by an identifying citation or number. The disclosures of these publications, patents and published patent application or granted patents are hereby incorporated by reference into the present disclosure to more fully describe the state of the art and field to which this present disclosure and intention pertains.

[0039]

[0033] As used herein, certain terms may have the following defined meanings unless stated otherwise.

[0040]

[0034] As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references or terms, unless the context clearly dictates otherwise.

[0041]

[0035] It is contemplated that the biopolymer may be soluble, insoluble or semi-soluble in water. The term “polymer”, “biopolymer”, “natural polymer”, “natural biopolymer” and other equivalent in meaning terms are used interchangeably and have the same meaning, unless stated expressly otherwise or it clear from the particular context that a different meaning is intended.

[0042]

[0036] As used herein, the term “insoluble biopolymer” refers to a biopolymer that is “insoluble” in a polar solvent such as water and this term encompasses similar terms for example “non-water-soluble”, “not soluble in water”, “water-insoluble”, or “indissoluble”. Solubility or lack of such as insolubility can typically be observed by a separation e.g. two separate phases in an aqueous environment, for instance biopolymer deposits / sediments at a bottom or floating at the top of the aqueous solution. As used herein, the term “semisoluble biopolymer” refers to a biopolymer that may be solubilised in a polar solvent such as water but under certain conditions for example heat, addition of chemicals such as organic acids and / or alcohols.

[0043]

[0037] For example, the term “organic acid” includes a single or plurality of organic acids, such as one or more organic acid selected from the group consisting of lactic acid, citric acid, acetic acid, linoleic acid, gluconic acid, glucuronic acid, formic acid, and butanoic acid. As used herein the term “organic acid” refers to a solution or solvent, which when added to or in the presence of a polymer such as a biopolymer e.g. natural biopolymer, can dissolve the biopolymer or has the capability of dissolving the biopolymer such as substantially dissolving the biopolymer. By way of example, organic acid refers to a solution or solvent, which when added to or in the presence of a biopolymer, has the capability to lead to an enhanced dissolution of the biopolymer. In some embodiments, the biopolymer is readily soluble in the organic acid. In some Attorney Reference No: 6666-P008-SAR-PCT embodiments the biopolymer is semi-soluble in the organic acid. In some embodiments, the one or more organic acid is selected from the group consisting of lactic acid, citric acid, acetic acid, linoleic acid, gluconic acid, glucuronic acid, formic acid, and butanoic acid. In some embodiments the organic acid is lactic acid.

[0044]

[0038] By way of a further example, the term “antisolvenf ’ includes a single or plurality of antisolvents such as one or more antisolvents selected from the group consisting of acetone, ethanol, methanol, propanol, isopropanol, butanol and water. As used herein the term “antisolvenf’ refers to a solvent in which a polymer such as a biopolymer e.g. natural biopolymer, has reduced solubility. By way of example, the term “antisolvenf’ refers to a solution or solvent, which when added to or in the presence of a biopolymer does not readily dissolve the biopolymer or has a lower capability or capacity of dissolving the biopolymer. In some embodiments the biopolymer is not readily dissolved in the antisolvent. In some embodiments the biopolymer is semi-soluble in the antisolvent. In some embodiments, the one or more antisolvents is selected from the group consisting of acetone, ethanol, methanol, propanol, isopropanol, butanol and water. In some embodiments, the antisolvent is ethanol. In some embodiments, the antisolvent is water. In some embodiments, the antisolvent is ethanol. In some embodiments of the present invention, the antisolvent is an alkali. In some embodiments of the present invention, there is provided a nanoparticle composition, wherein the nanoparticle composition further comprises an antisolvent. In some embodiments of the present invention, there is provided a nanoparticle composition for delivery of an active ingredient (Al), the composition comprises an organic acid, biopolymer, at least one Al and an antisolvent.

[0045]

[0039] As used herein, the term “biopolymer” refers to a polymer which can be produced by a cell, tissue or a living organism such as green plants, algae, crustaceans (e.g. crabs or shrimps), fungi, roots, tubers, leaves, petals, seeds fruits, and others or it may be sourced from nature. In some embodiments the biopolymer is a natural polymer. In some embodiments the biopolymer is 100% natural biopolymer. It is contemplated that the natural polymer consists of monomeric units that are covalently bonded to form larger polymerised molecules which can be branched such as heterogenous phenylpropanoid biopolymer with a three dimensionally branched architecture lignin or linear such as linear heteropolysaccharide chitosan. In the present invention, the biopolymer encompasses polypeptides, polysaccharides and polynucleotides molecules. By way of example, the biopolymer comprises one or more selected from the group consisting of lignin, chitin, chitosan, cellulose, starch, hemicellulose, amylose, actin, fibrin, collagen, fibroin and keratin or derivatives thereof. In some embodiments, the biopolymer comprises one or more selected from the group consisting of lignin, chitin, chitosan, cellulose, starch, hemicellulose, amylose, actin, fibrin, collagen, fibroin and keratin or derivatives thereof. Lignin is a class of complex organic polymers that form important structural materials in the support tissues of algae, vascular plants, including the bark, and herbaceous plants, such as wood (e.g. softwood and hardwood), cereals straw, cane bagasse, grass, linen, Attorney Reference No: 6666-P008-SAR-PCT jute, hemp, or cotton. Lignin can also have mineral source, such as peat, leonardite and coal. In its native form, chemically, lignin is a very irregular, randomly cross-linked polymer of phenylpropane units joined by many different linkages, with a weight average molecular weight of 20,000 Daltons or higher. In some embodiments of the present invention, the biopolymer is an essentially pure lignin. As used herein, the term “essentially pure lignin” refers to at least 80% pure lignin on a dry raw biomass basis, preferably at least 90% pure lignin, more preferably at least 95% pure lignin, the remainder being extractives and carbohydrates such as hemicelluloses as well as inorganic matter.

[0046]

[0040] It is intended by the present invention that the biopolymer can be a co-polymer. It is thus intended that the biopolymer is a co-polymer. As used herein the term “co-polymer” refers to a biopolymer comprising two or more different monomer units and / or two or more different polymers. The co-polymer can be comprised of polymers selected from two or more of lignin, chitin, chitosan, cellulose, starch, hemicellulose, amylose, actin, fibrin, collagen, fibroin and keratin, and combinations thereof or derivatives thereof. The present invention encompasses co-polymers including, but not limited to lignin + chitin, lignin + chitosan, chitin + cellulose, chitin + collagen, chitin + silk, chitosan + silk, chitosan + cellulose, chitosan + collagen, cellulose + collagen, cellulose + silk, collagen + silk and others. In some embodiments of the present invention, the biopolymer is a co-polymer of lignin and chitin. In some embodiments of the present invention, the biopolymer is a co-polymer of lignin and chitosan.

[0047]

[0041] The present inventors surprisingly and unexpectedly observed that when a polymer such as a biopolymer as described herein e.g. lignin, is suspended in a polar solvent such as water or alkali solvent and the resulting solution is mixed with an antisolvent solution containing a different polymer such as a biopolymer as described herein e.g. chitosan, it was possible to generate nanoparticle compositions comprising co-polymer with greater efficiency, stability of suspension, stability of nanoparticles, ease of encapsulation of an active ingredient, effective delivery and release of functional active ingredient at the target or site of administration. In other words, the combined and concomitant use of a solvent and an antisolvent which was counterintuitive was unexpectedly able to produce nanoparticle compositions with the above advantageous properties as described.

[0048]

[0042] All numbers or numerals as used herein that indicate amounts, ratios of materials, physical properties of materials, and / or use are to be understood as modified or qualified by the term "about," except as otherwise explicitly indicated.

[0049]

[0043] As used herein, the term "about" includes the recited number or number and + / - 10% from the recited numeral or number. By way of non-limiting example, the term "about ten (10)" would encompass nine (9) to eleven (11) or 9-11. Attorney Reference No: 6666-P008-SAR-PCT

[0050]

[0044] It is contemplated by the present invention that the nanoparticle composition may carry, transport and / or provide a nanocarrier for conveying, delivering and providing such as releasing an active ingredient of interest to a target. The target can be a subject, an area or a site such as agrarian land, farmed land, field or forest and others. The target can be aquaculture such as fish farming, crustacean farming such as lobsters, crabs, shrimps and barnacles, as well as mussel farming.

[0051]

[0045] As used herein, the term “subject” means any animal, such as a vertebrate, preferably a mammal such as human or animal, to whom will be or has been administered the nanoparticle composition of the present invention and according to embodiments of the invention and the examples. A subject, may be in need of or has been the object of observation or experiment of, treatment, prevention or management of a disease, condition or symptom. While the emphasis of the present disclosure resides with subjects, those of skill in the art will readily recognise that the present invention is also equally applicable and effective to non-human subjects (i.e. vertebrate animals) such as, for example, livestock (e.g. cattle, horses and sheep), exotic animals (e.g. pandas, big cats such as tigers, lions and pumas, elephants, bats and similar animals) and also companion animals (such as dogs, cats and rats).

[0052] 146] As used herein the term “active ingredient” or “Al” refers to a pharmaceutical, nutraceutical, probiotic, prebiotic, agrochemical or cosmetic compound, substance or cellular extract. As used herein the Al encompasses any compound, substance or cellular extract that can provide at least one beneficial effect or benefit. For instance, the Al may be a pharmaceutical compound, or a drug substance, a nutraceutical, a probiotic, a prebiotic, food supplement, an agrochemical such as a pesticide, herbicide, fungicide, or insecticide, a cosmetic compound, antioxidants, or cellular extract. In some embodiments of the present invention, the Al is one or more flavonoid selected from the group consisting of morin, naringenin, catechin, quercetin, epigallocatechin and others.

[0053]

[0047] As used herein the nanoparticle composition can carry, transport and / or provide a nanocarrier for conveying, delivering and providing such as releasing a pharmaceutical compound with pharmaceutical activity including, but not limited to, analgesics, anesthetics, anti-addiction agents, antibacterials, anticonvulsants, antidementia agents, antidepressants, antibodies, antiemetics, antifungals, antigout agents, anti-inflammatories, antimigraine agents, antioxidant agents, antimyasthenic agents, , antimycobacterials, antineoplastics, anti-obesity agents, antiparasitics, antipsychotics, antivirals, bipolar agents, blood glucose regulators, blood products, cardiovascular agents, central nervous system agents, contraceptives, dental and oral agents, dermatological agents, electrolytes, eczema agents, minerals, vitamins, gastrointestinal agents, genitourinary agents, hormonal agents, hormone suppressant, , immunological agents, infertility agents, inflammatory bowel disease agents, metabolic bone disease agents, ophthalmic agents, otic agents, Attorney Reference No: 6666-P008-SAR-PCT respiratory tract agents, sexual disorder agents, skeletal muscle relaxants, vaccines (mRNA, attenuated or inactivated), bioflavonoids such as morin, naringenin, catechin, quercetin, epigallocatechin and others.

[0054] H8] As used herein the nanoparticle composition can carry, transport and / or provide a nanocarrier for conveying, delivering and providing such as releasing a probiotic. As used herein the term “probiotic” refers to a bacterial strain such as lactic acid bacterial strain or isolate, including variants thereof, or extract therefrom or combination thereof, with the capability of exerting a beneficial effect on the subject to which they are administered or applied, preferably a beneficial effect on the health status. Probiotics are organisms, which when they are administered to a subject, especially but not exclusively as a food ingredient confer a health benefit to the host such as a subject. The beneficial effects may be achieved through interactions of the bacterial strain or combination thereof in the context of the invention, with the microbiota of the host to which they are administered or applied. Accordingly, the “probiotic” feature used as an adjective to qualify the bacterial strain, active ingredient, composition, and other food or compositions described herein, means that the same has the intended functionality encompassed by the “probiotic” definition.

[0055]

[0049] As used herein the nanoparticle composition can carry, transport and / or provide a nanocarrier for conveying, delivering and providing such as releasing an active ingredient selected from the group consisting of flavours, flavour precursors, aromas, aroma precursors, taste enhancers, salts, sugars, aminoacids, polysaccharides, enzymes, peptides, proteins or carbohydrates, food supplements, food additives, hormones, bacteria, plant extracts, medicaments, drugs, pharmaceuticals, bio-pharmaceuticals, biosimilars, vaccines, antigens, antibodies (Abs), peptibodies, antibody-drug-conjugates (ADCs), nutrients, chemicals for agro-chemical or cosmetic applications, carotenoids, vitamins, antioxidants or nutraceuticals selected from the group comprising of lutein, lutein esters, [beta] -carotene, tocopherol, tocopherol acetate, tocotrienol, lycopene, Co-QlO, flax seed oil, fish oil, omega-3 oils, omega-6 oils, DHA, EP A, arachidonic- rich oils, LCPUFA oils, menthol, mint oil, lipoic acid, vitamins, polyphenols and their glycosides, ester and / or sulfate conjugates, isoflavones, flavonols, flavanones and their glycosides such as hesperidin, flavan 3-ols comprising catechin monomers and their gallate esters such as epigallocatechin gallate and their procyanidin oligomers, vitamin C, vitamin C palmitate, vitamin A, vitamin B12, vitamin D, CC-and [gamma] -polyunsaturated fatty acids, phytosterols, esterified phytosterol, non-esterified phytosterol, zeaxanthine, caffeine and a combination thereof.

[0056] [50| As used herein, the term “treatment” or “treating” refers to an amelioration, prophylaxis, or reversal of a disease, condition, syndrome or disorder, or of at least one discernible symptom thereof. It is also contemplated that the treatment, as described herein throughout and based on data, leads to one or more of clinical improvement, reduction in the severity of disease in a patient. In some embodiments, “treatment” or “treating” refers to an amelioration, prophylaxis, or reversal of at least one measurable physical parameter Attorney Reference No: 6666-P008-SAR-PCT related to the disease, condition, syndrome or disorder being treated, not necessarily discernible in or by the subject. In some embodiments, “treatment” or “treating” refers to inhibiting or slowing the progression of a disease or disorder, either physically, e.g., stabilisation of a discernible symptom, physiologically, e.g., stabilisation of a physical parameter, or both. In some embodiments, “treatment” leads to partial or complete remission of the disease or disorder. In some embodiments of the present invention, treatment leads to a reduction by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of a disease, condition, syndrome or disorder being treated.

[0057]

[0051] The present inventors surprisingly observed that it was possible to synthesise biopolymer nanocarriers using less expensive, environmentally friendly solvents; faster and more sensitive processes requiring less equipment, more simple methods for their modification and characterization. The present inventors also surprisingly observed that it was possible to provide nanoparticle composition for delivery of active ingredients where the nanoparticles advantageously have tunable size and dimensions, high encapsulation capacity and sustainable release behavior. The present inventors further surprisingly and unexpectedly observed that it was possible to provide nanoparticle composition for delivery of active ingredients, which nanoparticle compositions can find application in various fields of biomedical sciences, pharmaceutical technology, agrochemical and food industries due to their experimentally proven antioxidant potential and antimicrobial activity.

[0058]

[0052] According to an aspect of the present invention, there is provided a nanoparticle composition for delivery of an active ingredient, the composition comprises an organic acid, biopolymer and at least one active ingredient.

[0059]

[0053] In some embodiments of the present invention, the organic acid comprises one or more selected from the group consisting of lactic acid, citric acid, acetic acid, linoleic acid, gluconic acid, glucuronic acid, formic acid, and butanoic acid. In some embodiments of the present invention, the organic acid is lactic acid.

[0060]

[0054] The skilled person would appreciate that different biopolymers would have different solubility in organic acid and be able to select the appropriate organic acid and concentration. In some embodiments of the present invention, the concentration of the organic acid comprises about 0.01% v / v of the composition, preferably about 0.05% v / v of the composition, preferably about 0.1% v / v of the composition, preferably about 0.2% of the composition, preferably about 0.4% of the composition preferably about 0.8% of the composition, preferably about 1% of the composition, preferably about 1.2% of the composition, preferably about 1.4% of the composition, preferably about 1.8% of the composition, preferably about 2% of the composition, preferably about 3% of the composition, preferably about 4% of the composition, preferably about 5% of the composition, preferably about 6% of the composition, preferably about 7% of the Attorney Reference No: 6666-P008-SAR-PCT composition, preferably about 8% of the composition, preferably about 9% of the composition, preferably about 10% of the composition, preferably about 11% of the composition, preferably about 12% of the composition, preferably about 13% of the composition, preferably about 14% of the composition preferably about 15% of the composition or more. In some embodiments, the organic acid comprises 1% lactic acid of the composition. In some embodiments, the biopolymer is dissolved in 1% lactic acid. In some embodiments, chitosan is dissolved in 1% lactic acid.

[0061]

[0055] In some embodiments of the present invention, there is provided a nanoparticle composition, wherein the biopolymer comprises one or more selected from the group consisting of lignin, chitin, chitosan, cellulose, starch, hemicellulose, amylose, actin, fibrin, collagen, fibroin and keratin. In some embodiments of the present invention, there is provided a nanoparticle composition, wherein the biopolymer is a copolymer. In some embodiments of the present invention, there is provided a nanoparticle composition, wherein the biopolymer is a co-polymer of lignin and chitin. In some embodiments of the present invention, there is provided a nanoparticle composition, wherein the biopolymer is a co-polymer of lignin and chitosan.

[0062]

[0056] In some embodiments of the present invention, there is provided a nanoparticle composition, wherein the concentration of the biopolymer is from about 100 mg / L to about 10000 mg / L, from about 300 mg / L to about 9000 mg / L, from about 600 mg / L to about 8000 mg / L, from about 900 mg / L to about 7000 mg / L, from about 1000 mg / L to about 6000 mg / L, from about 1300 mg / L to about 5000 mg / L.

[0063]

[0057] In some embodiments of the present invention, the concentration of biopolymer comprises about 0.1%, preferably about 0.15%, preferably about 0.2%, preferably about 0.25%, preferably about 0.3%, preferably about 0.35%, preferably about 0.4%, preferably about 0.5% of the composition, preferably about 1%, preferably about 1.25%, preferably about 1.5%, preferably about 2%, preferably about 3%, preferably about 4%, preferably about 5%, preferably about 6%, preferably about 7% of a solution or more.

[0064]

[0058] In some embodiments, 0.5% of biopolymer is dissolved in a 1% organic acid. In some embodiments, 0.5% the biopolymer chitosan is dissolved in a 1% lactic acid.

[0065]

[0059] In some embodiments of the present invention, there is provided a nanoparticle composition comprising at least one active ingredient, wherein at least one active ingredient is one or more selected from the group consisting of a pharmaceutical product or compound, an agrochemical product or compound, a phytochemical product or compound, a cosmetic substance, a food constituent or a dietary supplement.

[0066]

[0060] In some embodiments of the present invention, there is provided a nanoparticle composition wherein the organic acid, the biopolymer and the at least one active ingredient have been mechanically processed into a stable aqueous nanoparticle suspension. In some embodiments of the present invention, there is provided a nanoparticle composition wherein the organic acid, the biopolymer and the at least one Attorney Reference No: 6666-P008-SAR-PCT active ingredient have been subjected to homogenisation. In some embodiments of the present invention, mechanical processing involves shearing conditions and / or mechanical energy and / or homogenisation energy. In some embodiments of the present invention, mechanical processing involves high-shearing conditions and / or high mechanical energy. In embodiments the high-shearing conditions and / or high mechanical energy is obtained by a process including for example mechanical shearing, sheer thinning, planetary ball milling, ball milling, rolling mill, vibrating ball mill, tumbling stirred ball mill, horizontal media mill, colloid milling. As indicated hereinafter, the high-shearing conditions and / or high mechanical energy can be carried out for a duration, under parameters, under suitable conditions, until a desirable change of state is obtained, for example stability of the nanoparticles, change of turbidity, a change in viscosity, a change from a slurry to a paste, level of encapsulation of the active ingredient and others.

[0067] [611 In some embodiments of the present invention, the high-shearing conditions and / or high mechanical energy requires using a suitable device or apparatus. In some embodiments of the present invention the device or apparatus include for example a ball miller, a twin-screw extruder, a high-pressure homogenizer, a blade homogenizer, a stirring homogenizer, a disperser, a nanomaterial synthesis nebuliser, a rotorstator homogenizer, a high-shear mixer, a plowshare mixer, a dynamic mixer, a plough mixer, a turbine mixer, a speed mixer, an attrition miller, ultrasound irradiation, a sonicator, a cell lysor, a polytron, a ribbon agitator, a microfluidizer, a high pressure homogenizer, and combinations thereof. In some embodiments of the present invention the mechanical processing comprises ultrasound irradiation. In some embodiments of the present invention the mechanical processing comprises ultrasound homogenisation.

[0068] |62] In some embodiments of the present invention, there is provided a nanoparticle composition wherein the organic acid, the biopolymer and the at least one active ingredient following mechanical processing have been converted or processed into a stable aqueous nanoparticle suspension.

[0069]

[0063] In some embodiments of the present invention, there is provided a nanoparticle composition wherein the organic acid, the biopolymer and the at least one active ingredient following ultrasound homogenisation have been processed or converted into a stable aqueous nanoparticle suspension.

[0070] [64[ As used herein, the terms “stable suspension”, or similar terms that may be used herein interchangeably such as “homogenous suspension” or “stable biopolymer suspension” or simply “nanoparticle biopolymer suspension”, all refer to a suspension of insoluble and / or semi-soluble biopolymer particles such as co-polymer particles, that have been stably dispersed within a solution.

[0071]

[0065] Stability of the biopolymer suspensions may be assessed by any suitable means. In preferred embodiments, the stability is measured or observed by a lack of separation, for example, one single phase instead of two separate phases in a mixture, for instance absence of biopolymer deposits / sediments at a bottom or floating at the top of the mixture. Preferably, biopolymer suspensions in accordance with the Attorney Reference No: 6666-P008-SAR-PCT present invention are stable (e.g. absence of separation or ready leakage of encapsulated active ingredient) for at least 1 day, or at least 1 week, or at least one month, or at least one year or more.

[0072]

[0066] In some embodiments of the present invention, the stability of the nanoparticles is determined according to the level of encapsulation of an active ingredient of an active ingredient from the nanoparticles. In some embodiments of the present invention, the stability of the nanoparticle suspension is determined according to the level of encapsulation of an active ingredient or level of leakage of an active ingredient after nanoparticle formation such as following mechanical processing and / or homogenisation treatment

[0073]

[0067] In some embodiments, the stability of the nanoparticle suspension may be determined according to the concentration of non-encapsulated active ingredient retained in the suspension or the supernatant, after encapsulation or any further treatment.

[0074]

[0068] In some embodiments of the present invention, the concentration of the non-encapsulated active ingredient and / or leakage from the nanoparticles, such as for example flavonoid as an example active ingredient, can be determined spectrophotometrically using any spectrophotometer apparatus or equipment such as a UV / Vis spectrophotometer.

[0075]

[0069] Without wishing to be bound by theory, the encapsulation efficiency can be calculated using the following mathematical equation:

[0076] [721 In some embodiments of the present invention, the nanoparticle composition comprises biodegradable compounds. In some embodiments, the nanoparticle composition comprises biodegradable active ingredients.

[0077]

[0073] Those skilled in the art are aware that particle size measurements may vary according to the measurement method and the state of the particles (e.g., particles in a wet state are typically larger than the same particles in a dry state). Typically, the particles will be in a wet or suspended stage when measured by dynamic light scattering (DLS) and in a dry stage when measured by scanning electron microscopy (SEM).

[0078]

[0074] In some embodiments of the present invention, there is provided a nanoparticle composition where the nanoparticle composition comprises nanoparticles or spheres with an average diameter or size in the range of from about 10 nm to about 300 nm as measured by scanning electron microscopy (SEM).

[0079]

[0075] In some embodiments of the present invention there is provided a nanoparticle composition the composition comprising nanoparticles or spheres having an average diameter or size of about 20 nm to about 280 nm, or about 25 nm to about 285 nm, as measured by scanning electron microscopy (SEM). Attorney Reference No: 6666-P008-SAR-PCT

[0080]

[0076] In some embodiments of the present invention there is provided a nanoparticle composition, the composition comprising nanoparticles or spheres having an average diameter or size of about 30 nm to about 270 nm, or about 35 nm to about 275 nm, as measured by scanning electron microscopy (SEM).

[0081]

[0077] In some embodiments of the present invention there is provided a nanoparticle composition the composition comprising nanoparticles or spheres having an average diameter or size of about 40 nm to about 260 nm, or about 45 nm to about 265 nm, as measured by scanning electron microscopy (SEM).

[0082]

[0078] In some embodiments of the present invention there is provided a nanoparticle composition the composition comprising nanoparticles or spheres having an average diameter or size of about 50 nm to about 250 nm, or about 55 nm to about 255 nm, as measured by scanning electron microscopy (SEM).

[0083]

[0079] In some embodiments of the present invention there is provided a nanoparticle composition the composition comprising nanoparticles or spheres having an average diameter or size of about 60 nm to about 240 nm, or about 65 nm to about 245 nm, as measured by scanning electron microscopy (SEM).

[0084]

[0080] In some embodiments of the present invention there is provided a nanoparticle composition the composition comprising nanoparticles or spheres having an average diameter or size of about 70 nm to about 230 nm, or about 75 nm to about 235 nm, as measured by scanning electron microscopy (SEM).

[0085]

[0081] In some embodiments of the present invention there is provided a nanoparticle composition the composition comprising nanoparticles or spheres having an average diameter or size of about 80 nm to about 220 nm, or about 85vnm to about 220 nm, as measured by scanning electron microscopy (SEM).

[0086] [821 In some embodiments of the present invention there is provided a nanoparticle composition the composition comprising nanoparticles or spheres having an average diameter or size of about 90 nm to about 210 nm, or about 95 nm to about 215 nm, as measured by scanning electron microscopy (SEM).

[0087]

[0083] In some embodiments of the present invention there is provided a nanoparticle composition the composition comprising nanoparticles or spheres having an average diameter or size of about 100 nm to about 200 nm, or about 105 nm to about 205 nm, as measured by scanning electron microscopy (SEM).

[0088]

[0084] Accordingly, in an aspect of the present invention, there is provided a method for producing a co-polymer nanoparticle composition, the method comprising the steps of: a. supplying an aqueous solution comprising a soluble 1stbiopolymer; b. contacting the solution of step a. with an antisolvent solution to form a pre-mixture; c. to the pre-mixture adding organic acid dissolved 2ndbiopolymer to form a mixture; and Attorney Reference No: 6666-P008-SAR-PCT d. treating the mixture to produce a lst+2ndco-polymer nanoparticles suspension.

[0089]

[0085] In some embodiments of the present invention, there is provided a method for producing a lst+2ndco-polymer nanoparticle composition, wherein the step of treating the mixture comprises mechanical processing into a stable aqueous nanoparticle suspension. In some embodiments of the present invention there is provided a nanoparticle composition comprising a lst+2ndco-polymer nanoparticles wherein the nanoparticles are devoid of Al, empty or unloaded.

[0090] |86] It has surprisingly been observed by the present inventors that the devoid of Al, empty or unloaded nanoparticle compositions of the present invention display or are capable of scavenging activity.

[0091]

[0087] In some embodiments of the present invention, there is provided a method for producing a lst+2ndco-polymer nanoparticle composition, wherein the antisolvent solution further comprises at least one active ingredient.

[0092]

[0088] In an aspect of the present invention, there is provided a method for producing a biodegradable lignin-chitosan co-polymer nanoparticle composition, the method comprising the steps of: a. supplying an aqueous solution comprising lignin; b. contacting the solution of step a. with an antisolvent solution to form a pre-mixture; c. to the pre-mixture adding organic acid dissolved chitosan to form a mixture; and d. treating the mixture to produce lignin-chitosan nanoparticles suspension, wherein the antisolvent solution comprises at least one active ingredient.

[0093]

[0089] In some embodiments of the present invention, there is provided method for producing a biodegradable lignin-chitosan co-polymer nanoparticle composition, wherein the step of treating the mixture comprises mechanical processing into a stable aqueous nanoparticle suspension.

[0094] [90| In some embodiments of the present invention, there is provided method for producing a biodegradable lignin-chitosan co-polymer nanoparticle composition, wherein the aqueous nanoparticle suspension comprises lignin-chitosan nanoparticles, and wherein the lignin-chitosan nanoparticles contain the active ingredient.

[0095]

[0091] In some embodiments of the present invention, there is provided method for producing a biodegradable lignin-chitosan co-polymer nanoparticle composition, wherein the organic acid is one or more selected from the group consisting of lactic acid, citric acid, acetic acid, linoleic acid, gluconic acid, glucuronic acid, formic acid and butanoic acid. Attorney Reference No: 6666-P008-SAR-PCT

[0096]

[0092] In some embodiments of the present invention, there is provided method for producing a biodegradable lignin-chitosan co-polymer nanoparticle composition, wherein the antisolvent solution comprises one or more selected from the group consisting of acetone, ethanol, methanol, propanol, isopropanol, butanol and water.

[0097]

[0093] According to an aspect of the present invention, there is provided a biodegradable lignin- chitosan co-polymer nanoparticle composition comprising nanoparticles, nanocarriers or nano-spheres, with an average diameter in the range of from about 10 nm to about 300 run. In some embodiments of the present invention there is provided a biodegradable lignin-chitosan co-polymer nanoparticle composition comprising nanoparticles, nanocarriers or nano-spheres, having an average diameter or size of about 20 nm to about 280 nm, or about 25 nm to about 285 nm, as measured by scanning electron microscopy (SEM).

[0098]

[0094] In some embodiments of the present invention there is provided a biodegradable lignin-chitosan co-polymer nanoparticle composition comprising nanoparticles, nanocarriers or nano-spheres, having an average diameter or size of about 30 nm to about 270 nm, or about 35 nm to about 275 nm, as measured by scanning electron microscopy (SEM).

[0099]

[0095] In some embodiments of the present invention there is provided a biodegradable lignin-chitosan co-polymer nanoparticle composition comprising nanoparticles, nanocarriers or nano-spheres, having an average diameter or size of about 40 nm to about 260 nm, or about 45 nm to about 265 nm, as measured by scanning electron microscopy (SEM).

[0100]

[0096] In some embodiments of the present invention there is provided a biodegradable lignin-chitosan co-polymer nanoparticle composition comprising nanoparticles, nanocarriers or nano-spheres, having an average diameter or size of about 50 nm to about 250 nm, or about 55 nm to about 255 nm, as measured by scanning electron microscopy (SEM).

[0101]

[0097] The compositions of the present invention can be encapsulated. As used herein the term “encapsulated” refers to the compositions of the present invention which have undergone an encapsulation process such as a process by which the compositions of the present invention with encapsulated Al, are packaged within an outer shell material to offer protection against unfavourable environmental conditions and wherever required or deemed necessary as contemplated in the present invention, allowing for their controlled release under appropriate conditions in environment such as intestinal conditions or intestinal environment. Several methods are known in the art for encapsulation of compositions of the present invention with encapsulated Al according to the present invention, such as with non-limiting examples only, spray drying, extrusion, emulsion or phase separation, freeze drying, ionotropic gelation and others. By way of example, encapsulation technology usually allows for immobilisation and stabilisation of AIs such as enzymes, probiotics or extracts therefore, according to the present invention, within semipermeable and / or Attorney Reference No: 6666-P008-SAR-PCT biocompatible materials. Here the skilled person would be for example familiar with for example, Prado et al., 2020, Applied Microbiology and Biotechnology 104, pages 1993-2006.

[0102]

[0098] In some embodiments of the present invention, the nanoparticle compositions of the present invention, may further be dried by vacuum, freeze drying, or oven drying to form dried nanoparticle compositions.

[0103]

[0099] In an aspect of the present invention there is provided a method for delivery of at least one active ingredient, the method comprising the steps of: a. providing a nanoparticle composition or a biodegradable lignin-chitosan copolymer nanoparticle according to the present invention, b. formulating the nanoparticle composition or the biodegradable lignin-chitosan copolymer nanoparticle; and c. administering the formulated composition or biodegradable lignin-chitosan copolymer nanoparticle of step b..

[0104]

[0100] In some embodiments of the present invention there is provided a method for delivery of at least one active ingredient, wherein the formulated nanoparticle composition or the biodegradable lignin- chitosan co-polymer nanoparticle comprises at least one acceptable additive, palatant, excipient, binder, diluent, disintegrant, and / or adjuvant. In some embodiments of the present invention there is provided a method for delivery of at least one active ingredient, wherein the formulated nanoparticle composition is formulated for administering to a subject using one or more of the group of routes of administration consisting of per oral, intravenous, parenteral, rectal, nasal, topical (e.g., fransdermal and intraocular), intravesical, enteral, vaginal, transurethral, intradermal, aural, intralesional, endoscopical, transmucosal, sublingual and intestinal.

[0105] 1101] The nanoparticle compositions of the present invention when configured for drug delivery may be formulated as stable emulsions comprising fatty acids (e.g. C10-C22 fatty acids), an oil and / or a wax, and / or comprising N-Acetyl Glucosamine, and / or emulsifiers and preservatives, and / or comprising additives, including, but not limited to, preservatives, stabilizers and emulsifiers. In embodiments the additive is selected from Cetyl alcohol, Glyceryl stearate, Soy butter, PC90, Tara Gum, PSC3, PEG, Guar, Xanthan gum, Agarose, Sodium Hyaluronate, Tween 80™ and Glycerol. The additive(s) may be added prior, during and / or after combination of the biopolymer with the API.

[0106] [1021 As used herein the term “administration” should be understood to encompass for example delivery to a subject for example per oral, intravenous, parenteral, inhalation, pulmonary, rectal, nasal, topical (e.g., fransdermal and intraocular), intravesical, intrathecal, enteral, pulmonary, intralymphatic, Attorney Reference No: 6666-P008-SAR-PCT intracavital, vaginal, transurethral, intradermal, aural, intramammary, buccal, orthotopic, intratracheal, intralesional, percutaneous, endoscopical, transmucosal, sublingual, intestinal administration and combinations thereof.

[0107] |W3| Peroral Administration: The nanoparticle compositions or nanocarriers or nano-spheres of the invention can be formulated to take the form of tablets or capsules prepared by conventional means with one or more pharmaceutically acceptable carriers (e.g., excipients such as binding agents, fillers, lubricants and disintegrants).

[0108]

[0104] Parenteral Administration: The nanoparticle compositions or nanocarriers or nano-spheres of the present invention can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection can be presented in unit dosage form in ampoules or in multi-dose containers with an optional preservative added. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass, plastic or the like. The formulation can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain agents such as suspending, stabilizing and / or dispersing agents. For example, a parenteral preparation can be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent (e.g., as substance in 1,3 -butanediol solution). Some of the acceptable vehicles and solvents that can be employed include for example water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0109]

[0105] Controlled-Release Administration: Controlled-release (or sustained-release) preparations can be formulated to extend the activity of a substance and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the lactic acid bacteria or extract therefrom, and consequently affect the occurrence of any side effects.

[0110]

[0106] Controlled-release preparations can be designed to initially release an amount of the Al as contained in the nanoparticle compositions or nanocarriers or nano-spheres that produces the desired therapeutic effect, and gradually and continually release other amounts of the substance to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant levels in the body, the Al contained in the nanoparticle compositions or nanocarriers or nano-spheres can be released from the dosage form at a rate that will replace the amount of substance being metabolised and / or excreted from the body. The controlled-release of the Al can be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, and / or other physiological conditions or molecules.

[0111]

[0107] Controlled-release systems can include, for example, an infusion pump which can be used to administer the substance in a manner similar to that used for delivering insulin or chemotherapy to the body Attorney Reference No: 6666-P008-SAR-PCT generally, or to specific organs or tissues such as adipose tissues. Typically, using such a system, the substance is administered in combination with a biodegradable, biocompatible polymeric implant that releases the substance over a controlled period of time at a selected site. Example polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target such as adipose tissue, thus requiring only a fraction of a systemic dosage.

[0112] 1108] In some embodiments, the regimen of administration of the the Al as contained in the nanoparticle compositions or nanocarriers or nano-spheres is suitable for peroral administration.

[0113]

[0109] In some embodiments, the regimen of administration of the Al as contained in the nanoparticle compositions or nanocarriers or nano-spheres comprises one or more of peroral administration.

[0114] [HO] It will be appreciated by those skilled in the art that the amount or dose of the Al as delivered by the nanoparticle compositions or the biodegradable lignin-chitosan co-polymer nanoparticle compositions, as contemplated in the present invention, will vary with the nature or severity of the disease, syndrome or condition being treated; the type of the disease, syndrome or condition; the stage of the disease, syndrome or condition; the age of the subject; the weight and the overall condition of the subject, and will be ultimately at the discretion of the subject or wherever applicable at the recommendation of the attendant physician for example where the subject is on existing pharmaceutical therapy.

[0115] [H l] In some embodiments, the dose of the Al as delivered by the nanoparticle compositions described herein may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day or per administration.

[0116]

[0112] In some embodiments, the dose of Al as delivered by the nanoparticle compositions described herein per administration will typically be in the range of about 0. 1 to 2000 mg / kg of body weight, about 0. 15 to 1750 mg / kg of body weight, about 0.2 to 1700 mg / kg of body weight, about 0.3 to 1500 mg / kg of body weight, about 0.5 to 1250 mg / kg of body weight, about 1 to 1000 mg / kg of body weight, about 2 to 900 mg / kg of body weight, about 3 to 800 mg / kg of body weight, about 4 to 700 mg / kg of body weight, about 5 to 600 mg / kg of body weight, about 10 to 500 mg / kg of body weight, administered as one, two, three, four or more doses or sub-doses per day or per administration.

[0117] 1113] In some embodiments, the dose is 400 mg / kg of body weight administered as one, three, four or more doses or sub-doses per day or per administration.

[0118]

[0114] For the avoidance of doubt, the skilled person will understand that references to certain maximum amounts and concentrations in plasma may also require a minimum of a therapeutically effective amount in the plasma. Attorney Reference No: 6666-P008-SAR-PCT

[0119]

[0115] The skilled person will understand that references to certain maximum (i.e. where values are indicated as being “below”) and minimum (i.e. where values are indicated as being “at least”) amount and / or concentrations in plasma may be combined to form ranges (i.e. wherein the amount in plasma is in a range that is from the minimum value to the maximum value).

[0120]

[0116] As used herein, the term “significant” when referring to for example reducing, enhancing, remission, amelioration, prophylaxis, or reversal, that is statistically significant, not due to chance alone, which has a p-value of 0.05 or less. In particular, the term “significant” can have a p-value of less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc., when referring to for example reducing, enhancing, remission, amelioration, prophylaxis, weight loss, size of white adipose cells, intracellular lipid deposition, increase in brown adipose cells or glucose uptake, regression, or reversal of disease, disorder or symptom caused by or associated with lipid metabolism such as abnormal lipid metabolism for example when compared with the level or biomarker determinations in one or more non-treated patients or when compared with the level or biomarker determinations in the same patient observed at a different time point such as an earlier time point (e. g. comparison with a "base line" level or placebo). Those of skill in the relevant art would be familiar with different statistical calculation approaches, examples include, t-test, z-test, sample test, O’Brien- Fleming method for normally distributed data etc. Reference here is also made to the Figures as described and incorporated in the present invention.

[0121]

[0117] In some embodiments of the present invention there is provided a method for delivery of at least one active ingredient, wherein the formulated nanoparticle composition is formulated for agricultural administration using one or more of the group of routes of administration consisting of hydraulic spraying, backpack spraying, basal trunk spraying, aerial spraying, injection, soil injection, soil incorporation, ropewick and wiper treatment.

[0122]

[0118] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples.

[0123] 1119] MATERIALS, METHODS AND EXPERIMENTAL EXAMPLES

[0124]

[0120] I. Synthesis of lignin / chitosan nanoparticles

[0125]

[0121] Unloaded and flavonoid-encapsulated lignin / chitosan nanoparticles were synthesized by a combined solvent / antisolvent precipitation, self-assembly and sonication methodology.

[0126]

[0122] STAGE 1. Synthesis of lignin / chitosan nanoparticles suspension

[0127]

[0123] Prepare 25 mL aqueous solution of alkaline lignin with a concentration of 5000 mg / L, to which 1 mL of a 96% ethanol solution is slowly added dropwise while stirring on a magnetic stirrer for 3 min. The procedure is followed by the addition of 1 mL 0.5% chitosan solution in 1% lactic acid at a flow Attorney Reference No: 6666-P008-SAR-PCT rate of 0.5 mL / min. Other flow rates may also be employed such as slower rate such as 0.4 mL / min, 0.3 mL / min, 0.2 mL / min, 0.1 mL / min or slower. The synthesis procedure of the flavonoid-encapsulated nanoparticles follows the same steps, including the addition of 20 mg of flavonoid (morin, naringenin, catechin, quercetin, epigallocatechin) dissolved in 96% ethyl alcohol to the aqueous solution of alkaline lignin prior to the addition of the chitosan solution in lactic acid.

[0128] [1241 STAGE 2. Ultracentrifugation and washing of the nanoparticles

[0129] |125] The resulting suspension was subjected to a three-fold cycle, including stirring at 500 rpm for 30 min on a magnetic stirrer, subsequent transfer of the suspension to Eppendorf tubes, ultracentrifugation at 15,000xg at temperature T = 10°C and washing the nanoparticles with ultrapure water.

[0130] |126] STAGE 2.1. Spectrophotometric determination of the concentration of nonencapsulated flavonoid in the supernatant

[0131]

[0127] In the synthesis of flavonoid-encapsulated lignin / chitosan nanoparticles, the concentration of the non-encapsulated flavonoid in the supernatant after the first cycle of centrifugation before washing with ultrapure water was determined spectrophotometrically on a UV / Vis spectrophotometer at the corresponding maximum wavelength of absorption.

[0132]

[0128] STAGE 3. Ultrasonic homogenization

[0133]

[0129] The third stage of the synthesis technique involved ultrasonic homogenization of the nanoparticle suspension on an ultrasonic homogenizer in an ice bath in two cycles, each lasting 4 min at intensity of ultrasound irradiation 93-96%.

[0134]

[0130] STAGE 4. Lyophilization of the nanoparticles

[0135]

[0131] The homogenized particles were subjected to subsequent lyophilization in a vacuum lyophilizer at a temperature of T = -64°C. Figure 6.

[0136] 1132] II. Characterization of lignin / chitosan- nanoparticles

[0137]

[0133] STAGE 5. Physicochemical characterization of the nanoparticles

[0138]

[0134] • TEM analyses'. Nanoparticle suspensions were stained with 1% uranyl acetate in 70% methanol and placed on electron microscopy membranes pre-coated with a thin layer of formvar membrane. TEM analyzes were performed at high resolution using a transmission electron microscope (Figure 1).

[0139]

[0135] • FTIR analyses'. FTIR spectra of the pure biopolymers lignin and chitosan, flavonoids and nanoparticles (Figure 2) were obtained by disk technique with potassium bromide (KBr) in the wavelength range 400-4000 cm1on a FTIR spectrometer. Attorney Reference No: 6666-P008-SAR-PCT

[0140]

[0136] • The size and ^-potential of the nanoparticles are determined on a zeta analyzer (Table 1).

[0141] Table 1. Size and ^-potential of lignin / chitosan and morin-encapsulated lignin / chitosan nanoparticles.

[0142]

[0138] • The concentration of surface acid functional groups is determined by potentiometric titration (Figure 3).

[0143]

[0139] STAGE 6. In vitro biological activity of nanoparticles

[0144]

[0140] Antioxidant activity

[0145]

[0141] The DPPH and ABTS spectrophotometric assays are applied for determination of the scavenging activity of unloaded and loaded nanoparticles. Figure 4 presents the DPPH radical-scavenging activity of unloaded, morin-encapsulated lignin-chitosan nanoparticles, pure chitosan and morin at 1 min and 30 min.

[0146]

[0142] Antibacterial activity

[0147] 1143] The antimicrobial activity of both types of nanoparticles against bacterial strains Staphylococus aureus ATCC 25923, B. cercus. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 was determined by the micro-broth dilution method as described in the Clinical and Laboratory Standards Institute (CLSI).

[0148]

[0144] Minimum Inhibitory Concentrations (MICs) were defined as the lowest concentrations of the tested compounds resulting in optical density OD value close to negative controls (no growth). The assays were performed in triplicate with three independent experiments.

[0149]

[0145] The MICs of a three-component lignin-chitosan-morin mixture and morin-encapsulated lignin / chitosan nanoparticles against the four tested bacterial strains is presented in Table 2.

[0150]

[0146] Table 2. Minimum inhibitory concentrations (MICs) of a three-component lignin-chitosan- morin mixture and morin-encapsulated lignin / chitosan nanoparticles against S. aureus, B. cereus, E. coli and Ps. Aeruginosa strains. Attorney Reference No: 6666-P008-SAR-PCT

[0151]

[0147] STAGE 7. In vitro release of the encapsulated flavonoid in a simulated physiological environment

[0152] 1148] Experiments investigating the kinetics of in vitro release of encapsulated biologically active substances were carried out in a glass batch reactor equipped with a mechanical stirrer. The morin-loaded nanoparticles were stirred in 50 mL of enzyme-free simulated gastrointestinal physiological medium at pH = 6.5 and pH = 1.2 at temperature T = 37±0.5°C in a digital water bath. Samples were taken at predetermined time intervals. Morin concentrations in a simulated physiological environment were determined on a U V / Vis spectrophotometer equipped with 10 mm quartz cuvettes in the visible region of the spectrum at X = 390 nm. Volume corrections in the processing of experimental results are performed by replacing the withdrawn probe with an equivalent volume of fresh simulated gastrointestinal medium.

[0153]

[0149] The aim is to avoid saturation of the remaining solution. All experiments were performed in triplicate and the average values were taken to minimize random error. Flavonoid-free blanks and replicates at each release point were used for all experimental runs.

[0154]

[0150] The in vitro release kinetic curves of morin from morin-encapsulated lignin-chitosan nanoparticles in both simulated physiological compartments are presented in Figure 5.

[0155] 1151] In Figure 6, there is depicted a scheme of the methodical and experimental protocols for the synthesis and characterization of lignin / chitosan nanoparticles described herein and as claimed in the present invention.

[0156]

[0152] Advantages of the present invention

[0157]

[0153] In order to overcome the shortcomings and the challenges associated with the need for new methodologies for the synthesis of nanocarriers using less expensive, environmentally friendly solvents, faster and more sensitive processes requiring less equipment, more simple methods for their modification and characterisation, the present invention applies for the first time a new efficient green method for the Attorney Reference No: 6666-P008-SAR-PCT production and characterization of functional lignin / chitosan nanocarriers with tunable size, high encapsulation capacity and sustainable in vitro release behavior, which can find application in various fields of biomedical sciences, pharmaceutical technology and food industry due to their proven antioxidant potential and antimicrobial activity.

[0158]

[0154] The presented approach is substantiated by the fact that for the first time double adjuvant nanoparticles comprised of the linear heteropolysaccharide chitosan and the heterogenous phenylpropanoid biopolymer with a three dimensionally branched architecture lignin are going to be designed and intended for application as technological platforms for efficient encapsulation, increased bioavailability and sustained delivery of bioactive phytocompounds, nutraceuticals and / or drugs. Lignin and chitosan demonstrate favorable self compatibility offering versatility in blended formulations to meet specific requirements. Additionally, the biodegradability of both natural polymers, combined with their renewable origin and various active functional groups, contribute to the appeal in applications aimed at sustainable practices in the pharmaceutical, food industry and other areas. The ease of synthesis, the biocompatibility of novel nanoparticles, as well as the possibility for customisation of the current protocol, represent the major advantages of the presented methodology.

[0159]

[0155] The present invention provides simpler, safer and improved, for instance, in terms of physicochemical characteristics nano-formulations, characterised with better dispersibility, adhesive properties, improved encapsulation efficiency, sustained and predictable release of the encapsulated active compounds and enhanced biological activities such as biocompatibility, biodegradability, satisfactory antimicrobial and antioxidant properties.

[0160]

[0156] The disclosure illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognised that various modifications are possible within the scope of the disclosure claimed. It will also be appreciated that the method(s), use(s), compositions(s), combinations(s) and / or administrations(s) may be subject to numerous rearrangements, modifications and substitutions without departing from the scope of the present disclosure as set forth and defined by the following claims. Attorney Reference No: 6666-P008-SAR-PCT

[0161] REFERENCES:

[0162] Ali, K. et al. (2020). Role of Solvent System in Green Synthesis of Nanoparticles. In: Saquib, Q., Faisal, M., Al-Khedhairy, A. A., Alatar, A. A. (eds) Green Synthesis of Nanoparticles: Applications and Prospects . Springer, Singapore, https: / / doi.org / 10.1007 / 978-981-15-5179-6 3

[0163] Worku, L.A., Bachheti, R.K., Tadesse, M.G., Bachheti, A., Ali, D., Kumar, G., Chaubey, K.K., Juyal, A., Almarzoug, M.H.A. Synthesis of lignin nanoparticles from Oxytenanthera abyssinica by nanoprecipitation method followed by ultrasonication for the nanocomposite application. Journal of King Saud University - Science, 35(7), 2023, 102793, https: / / doi.org / 10, 1016 / i,iksus.2023. 102793

[0164] Mishra, P.K.; Ekielski, A. A Simple Method to Synthesize Lignin Nanoparticles. Colloids Interfaces 2019, 3, 52. htps: / / doi.org / 10.3390 / colloids3020Q52

[0165] Qian, Y.; Deng, Y.; Qiu, X.; Li, H.; Yang, D. Formation of uniform colloidal spheres from lignin, a renewable resource recovered from pulping spent liquor. Green Chem. 2014, 16, 2156-2163. doi: 10.3390 / polyml2112471.

[0166] Tardy, B.L., Richardson, J. J., Guo, J., Lehtonen, J., Agoa, M., Rojas, O.J., Lignin nano- and microparticles as template for nanostructured materials: formation of hollow metal -phenolic capsules. Green Chem., 2018, 20, 1335-1344. https: / / doi.org / 10.1039 / C8GC00Q64F

[0167] Tse, H. Y., Yeung, C. S., Lau, C. Y., Cheung, M. Y., Guan, J. Y., Islam, M. K., et al. (2022). One-pot synthesis to prepare lignin / photoacid nanohybrids for multifunctional biosensors and photo-triggered singlet oxygen generation. Green Chemistry, 24(7), 2904-2918. http: / / doi.org / 10. 1039 / d2gc00196a

Claims

Attorney Reference No: 6666-P008-SAR-PCTWHAT IS CLAIMED IS:

1. A nanoparticle composition for delivery of an active ingredient, the composition comprises an organic acid, biopolymer and at least one active ingredient.

2. A nanoparticle composition according to claim 1, wherein the organic acid comprises one or more selected from the group consisting of lactic acid, citric acid, acetic acid, linoleic acid, gluconic acid, glucuronic acid, formic acid, and butanoic acid.

3. A nanoparticle composition according to claim 1 or claim 2, wherein the organic acid is lactic acid.

4. A nanoparticle composition according to anyone of the preceding claims, wherein the concentration of the organic acid comprises about 0.01% v / v of the composition, preferably about 0.05% v / v of the composition, preferably about 0.1% v / v of the composition, preferably about 0.2% of the composition, preferably about 0.4% of the composition preferably about 0.8% of the composition, preferably about 1% of the composition, preferably about 1.2% of the composition, preferably about 1.4% of the composition, preferably about 1.8% of the composition, preferably about 2% of the composition, preferably about 3% of the composition, preferably about 4% of the composition, preferably about 5% of the composition, preferably about 6% of the composition, preferably about 7% of the composition, preferably about 8% of the composition, preferably about 9% of the composition, preferably about 10% of the composition, preferably about 11% of the composition, preferably about 12% of the composition, preferably about 13% of the composition, preferably about 14% of the composition preferably about 15% of the composition or more.

5. A nanoparticle composition according to anyone of the preceding claims, wherein the biopolymer comprises one or more selected from the group consisting of lignin, chitin, chitosan, cellulose, starch, hemicellulose, amylose, actin, fibrin, collagen, fibroin and keratin.

6. A nanoparticle composition according to claim 5, wherein the biopolymer is a co-polymer of lignin and chitin (lignin + chitosan).

7. A nanoparticle composition according to anyone of claims 5 or 6, wherein the concentration of the biopolymer is from about 100 mg / L to about 10000 mg / L, from about 300 mg / L to about 9000 mg / L, from about 600 mg / L to about 8000 mg / L, from about 900 mg / L to about 7000 mg / L, from about 1000 mg / L to about 6000 mg / L, from about 1300 mg / L to about 5000 mg / L.

8. A nanoparticle composition according to anyone of the preceding claims, wherein at least one active ingredient is one or more selected from the group consisting of a pharmaceutical product orAttorney Reference No: 6666-P008-SAR-PCT compound, an agrochemical product or compound, a phytochemical product or compound, a cosmetic substance, a food constituent or a dietary supplement.

9. A nanoparticle composition according to anyone of the preceding claims, wherein the organic acid, the biopolymer and the at least one active ingredient have been mechanically processed into a stable aqueous nanoparticle suspension.

10. A nanoparticle composition according to claim 9, wherein the organic acid, the biopolymers and the at least one active ingredient are biodegradable.

11. A nanoparticle composition according to claim 10, wherein the nanoparticle composition comprises biodegradable compounds.

12. A nanoparticle composition according to claim 11, wherein the nanoparticles have a diameter in the range of from about 10 nm to about 300 nm.

13. A method for producing a biodegradable lignin-chitosan co-polymer nanoparticle composition, the method comprising the steps of: a. supplying an aqueous solution comprising lignin; b. contacting the solution of step a. with an antisolvent solution to form a pre-mixture; c. to the pre-mixture adding organic acid dissolved chitosan to form a mixture; and d. treating the mixture to produce lignin-chitosan nanoparticles suspension, wherein the antisolvent solution comprises at least one active ingredient.

14. A method for producing a biodegradable lignin-chitosan co-polymer nanoparticle composition according to claim 13, wherein the step of treating the mixture comprises mechanical processing into a stable aqueous nanoparticle suspension.

15. A method for producing a biodegradable lignin-chitosan co-polymer nanoparticle composition according to claim 14, wherein the aqueous nanoparticle suspension comprises lignin-chitosan nanoparticles, and wherein the lignin-chitosan nanoparticles contain the active ingredient.

16. A method for producing a biodegradable lignin-chitosan co-polymer nanoparticles composition according to anyone of claims 13 to 15, wherein the organic acid is one or more selected from the group consisting of lactic acid, citric acid, acetic acid, linoleic acid, gluconic acid, glucuronic acid, formic acid and butanoic acid.

17. A method for producing a biodegradable lignin-chitosan co-polymer nanoparticles composition according to anyone of claims 13 to 16, wherein the antisolvent solution comprises one or moreAttorney Reference No: 6666-P008-SAR-PCT selected from the group consisting of acetone, ethanol, methanol, propanol, isopropanol, butanol and water.

18. A biodegradable lignin-chitosan co-polymer nanoparticle composition comprising nanoparticles with a diameter in the range of about 10 nm to about 300 nm prepared by the method of anyone of claims 13 to 17.

19. A method for delivery of at least one active ingredient, the method comprising the steps of: a. providing a nanoparticle composition according to anyone of claims 1 to 12 or claim 18; b. formulating the nanoparticle composition; and c. administering the formulated composition of step b..

20. A method according to claim 19, wherein the formulated nanoparticle composition comprises at least one acceptable additive, palatant, excipient, binder, diluent, disintegrant, and / or adjuvant.

21. A method according to claim 19 or claim 20, wherein the nanoparticle composition is formulated for administering to a subject using one or more of the group of routes of administration consisting of per oral, intravenous, parenteral, rectal, nasal, topical (e.g., transdermal and intraocular), intravesical, enteral, vaginal, transurethral, intradermal, aural, intralesional, endoscopical, transmucosal, sublingual and intestinal.

22. A method according to claim 19 or claim 20, wherein the nanoparticle composition is formulated for agricultural administration using one or more of the group of routes of administration consisting of hydraulic spraying, backpack spraying, basal trunk spraying, aerial spraying, injection, soil injection, soil incorporation, rope-wick and wiper treatment.

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