Micronized cross-linked hyaluronic acid compositions and its application in enhancing biological activities and rejuvenating tisuses

Abstract

Micronized cross-linked hyaluronic acid (HA) compositions are provided, comprising micronized cross-linked HA, a salt thereof, or a derivative thereof, wherein the micronized composition retains key structural properties of the originating cross-linked HA and exhibits inherent stability in an acceptable carrier without a separate surface-stabilizing agent. Pharmaceutical, dermatological, and cosmetic compositions comprising the micronized cross-linked HA, and methods of treating conditions or enhancing the appearance or feel of skin, hair, or nails, are also disclosed.

Description

MICRONIZED CROSS-LINKED HYALURONIC ACID COMPOSITIONS AND ITS APPLICATION IN ENHANCING BIOLOGICAL ACTIVITIES AND REJUVENATING TISUSES FIELD OF THE INVENTION

[0001] The present invention is in the field of cross-linked hyaluronic acid and uses thereof.BACKGROUND OF THE INVENTION

[0002] Cross-linked hyaluronic acid (HA) is a modified form of HA in which molecules are chemically bonded to create a stable, long-lasting structure. Compared to non-cross-linked HA, it offers several advantages, including longer durability, better volume retention, slower absorption, and enhanced hydration. These properties make it ideal for long-term applications.

[0003] Today, cross-linked HA is widely used in both cosmetic and medical fields. In cosmetic applications, it is primarily utilized in dermal fillers to restore volume, smooth wrinkles, and enhance facial contours. It also plays a role in skin boosters, which improve hydration and elasticity for a youthful appearance. In medical applications, cross-linked HA is employed in orthopedic injections for joint lubrication and osteoarthritis treatment, ophthalmology procedures such as eye surgery and dry eye treatment, and wound healing therapies. The structural stability of cross-linked HA makes it particularly effective in these medical treatments, providing sustained benefits overtime.

[0004] However, one of the key challenges associated with cross-linked HA is its limited penetration into tissues due to its dense and rigid structure. This reduced permeability can hinder its effectiveness in applications requiring deep tissue integration and hydration, potentially limiting its therapeutic potential. The inability to adequately diffuse into surrounding tissues may result in suboptimal performance, especially in cases where uniform distribution is critical. Therefore, there is an imminent need to enhance the penetrability of cross-linked HA while maintaining its structural integrity and desirable properties. Improving its permeability could expand its applications, increase its bioavailability, and ultimately enhance patient outcomes across various medical and cosmetic treatments.SUMMARY OF THE INVENTION

[0005] It is hence an object of the invention to disclose a micronized cross-linked hyaluronic acid (HA) composition, comprising cross-linked HA, a salt thereof, or a derivative thereof;wherein the micronized cross-linked HA composition retains key structural properties of the cross-linked HA it originates from.

[0006] In some embodiments, the micronized cross-linked HA exhibits inherent stability or maintains its stability within the acceptable carrier without dependence on any separate surface-stabilizing agent.

[0007] It is hence an object of the invention to disclose a micronized cross-linked hyaluronic acid (HA) composition, comprising cross-linked HA, a salt thereof, or a derivative thereof, including any combination thereof; wherein the micronized cross-linked HA composition (i) retains key structural properties of the cross-linked HA it originates from; and (ii) exhibits inherent stability or maintains its stability within the acceptable carrier without dependence on any separate surface-stabilizing agent.

[0008] In some embodiments, the micronized cross-linked HA composition is characterized by an average particle size of between 105 nm and 500 nm.

[0009] In some embodiments, the derivative thereof is selected from the group consisting of butanediol glycidyl ether cross-linked HA, divinyl sulfone cross-linked HA, polyethylene glycol diacrylate cross-linked HA, thiol-ene cross-linked HA, or carbodiimide cross-linked HA, including any combination thereof.

[0010] In some embodiments, the micronized cross-linked HA composition is characterized by a reduction of between about 50% and 95% in average particle size of macromolecule from which it originates.[Oil] In some embodiments, the micronized cross-linked HA composition is characterized by a hygroscopy that is at least 1.5 times greater than that of the cross-linked HA it originates from.

[0012] In some embodiments, the micronized cross-linked HA composition further comprises a pharmaceutical carrier.

[0013] In some embodiments, the composition is a pharmaceutical composition.

[0014] In some embodiments, the pharmaceutical composition is a dermatological composition.

[0015] In some embodiments, the pharmaceutical composition is for use in treatment or prevention of a disease, a disorder or a condition.

[0016] In some embodiments, the micronized cross-linked HA composition further comprises a cosmetic acceptable carrier.

[0017] In some embodiments, the composition is a cosmetic composition.

[0018] In some embodiments, the cosmetic is for use enhancing appearance or feel of skin, hair, or nails, including any combination thereof.

[0019] In some embodiments, the cosmetic composition is characterized by an at least penetration to a stratum comeum.

[0020] In some embodiments, the micronized cross-linked HA composition is characterized by enhanced permeability compared to the cross-linked HA composition it originates from.

[0021] In some embodiments, the micronized cross-linked HA composition further comprising an additive selected from the group consisting of food supplement, antioxidants fragrance, flavor, preservatives, colorants, emulsifiers, sweeteners, thickeners, stabilizers, acidulants, humectants, chelating agents, nutraceuticals, probiotics or prebiotics, including any combination thereof.

[0022] It is hence another object of the invention to disclose a pharmaceutical composition comprising (i) micronized cross-linked HA, a salt thereof, or a derivative thereof, including any combination thereof, as disclosed therein; and (ii) a pharmaceutical acceptable carrier; wherein the micronized cross-linked HA composition retains key structural properties of cross-linked HA it originates from.

[0023] In some embodiments, the pharmaceutical composition is for use in the treatment of a disease, a disorder, or a condition.

[0024] In some embodiments, the disease, disorder, or condition is selected from the group consisting of joint disorders, wound healing, inflammation and autoimmune conditions, or skin and hair health, including any combination thereof.

[0025] In some embodiments, the pharmaceutical composition is a dermatological composition.

[0026] It is hence another object of the invention to disclose a dermatologic composition comprising (i) micronized cross-linked HA, a salt thereof, or a derivative thereof, including any combination thereof; and (ii) a dermatologic acceptable carrier; wherein the micronized crosslinked HA composition retains key structural properties of cross-linked HA it originates from.

[0027] It is hence another object of the invention to disclose a cosmetic composition comprising (i) micronized cross-linked HA, a salt thereof, or a derivative thereof, including any combination thereof; and (ii) a cosmetic acceptable carrier; wherein the micronized cross-linked HA composition retains key structural properties of cross-linked HA it originates from.

[0028] In some embodiments, the cosmetic composition is for enhancing appearance or feel of skin, hair, or nails, including any combination thereof.

[0029] In some embodiments, enhancing appearance or feel is selected from the group consisting of improve fine line and wrinkles, promote skin rejuvenation, improve damaged hair, or strengthen nail, including any combination thereof.

[0030] In some embodiments, the cosmetic composition is characterized by penetration into the skin of at least a stratum comeum.

[0031] It is hence another object of the invention to disclose a method for treating adisorder, disease, or condition in a subject in need thereof, the method comprising contacting the subject's skin with a therapeutic effective amount of a pharmaceutical composition comprising (i) micronized cross-linked HA composition, a salt thereof, a derivative thereof, or any combination thereof, as disclosed herein; and (ii) a pharmaceutical acceptable carrier; wherein the micronized cross-linked HA composition retains key structural properties of cross-linked HA it originates from.

[0032] In some embodiments, the therapeutic effective amount is an amount sufficient to reduce, relieve, or prevent, including at least one symptom of the disorder, disease, or condition.

[0033] In some embodiments, the contacting is at least once a day for at least one consecutive day.

[0034] In some embodiments, the disease is selected from the group consisting of rheumatoid arthritis, atopic eczema, psoriasis, atopic dermatitis, or any inflammation of a skin.

[0035] It is hence another object of the invention to disclose a method for enhancing appearance or feel of skin, hair, or nails, including any combination thereof, in a subject in need, the method comprising contacting the subject's skin with an effective amount of a cosmetic composition comprising (i) micronized cross-linked hyaluronic acid (HA) composition, a salt thereof, a derivative thereof, or any combination thereof, as disclosed herein; and (ii) a cosmetically acceptable carrier; wherein the micronized cross-linked HA composition retains key structural properties of the cross-linked HA it originates from.

[0036] In some embodiments, the effective amount is an amount sufficient to enhance appearance or feel of skin, hair, or nails, including any combination thereof.

[0037] In some embodiments, the enhancing appearance or feel of skin, hair, or nails is selected from the group consisting of increase brightness, increase smoothness, increase plumpness, increase skin tone evenness, increase moisture, repair hair damage, prevent breakage, prevent peeling, reduce pigmentation, reduce wrinkles, including any combination thereof.

[0038] In some embodiments, the contacting is at least once a day for at least one consecutive day.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The presently disclosed subject matter may be more clearly understood upon reading the following detailed description of non-limiting exemplary embodiments thereof, with reference to the drawings.

[0040] Dimensions of components and features shown in the figures are chosen for convenience or clarity of presentation and are not necessarily shown to scale. Wherever possible, the same reference numbers will be used throughout the drawings and the following description to refer to the same and like parts:

[0041] Fig. 1 shows graphs presenting Fourier Transform Infrared of cross-linked HA composition before and after miniaturization;

[0042] Fig. 2 is a scanning electron microscopy image, presenting the micronized cross-linked HA composition, according to some embodiments of the present invention;

[0043] Figs. 3A-3B are images showing a subject left foot (3A) before and (3B) after applying a dermatological composition according to some embodiments of the present invention;

[0044] Figs. 4A-4B are Visia images comparing a cosmetic composition according to some embodiments of the present invention comprising (4 A) micronized cross-linked HA of the present invention versus a composition having micronized HA based on the same formula as 4A; and

[0045] Fig. 5 is a graph comparing the antioxidant capability of a micronized cross-linked HA compared to micronized HA.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the inventionpertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0047] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0048] According to one aspect of the present invention, there is provided a micronized cross-linked hyaluronic acid (HA) composition, comprising cross-linked HA, a salt thereof, or a derivative thereof; wherein the micronized cross-linked HA composition retains the key structural properties of the cross-linked HA it originates from.

[0049] As used herein, the term "cross-linked" refers to the formation of an at least one covalent bond between HA chains, creating a three-dimensional network that enhances the material's strength, thermal resistance, and chemical stability.

[0050] As used herein, the term "derivative" refers to a chemically modified cross-linked HA.Typically, a derivative is often formed by substituting a specific functional group within a molecule, for example, replacing a hydrogen atom with a hydroxyl group to form an alcohol. Non-limiting examples of cross-linked HA derivatives include but are not limited to butanediol glycidyl ether cross-linked HA, divinyl sulfone cross-linked HA, polyethylene glycol diacrylate cross-linked HA, thiol-ene cross-linked HA, or carbodiimides cross-linked HA, including any combination thereof.

[0051] As used herein, the terms "composition" and "micronized cross-linked HA" are used interchangeably and refer to a downsized version of the macromolecule from which it originates. This downsized version is characterized by a reduction in size while retaining the key structural properties of the macromolecule it originates from. As used herein, the term "downsized version" refers to an average particle size of between 105 nm and 500 nm.

[0052] The micronized cross-linked HA consists essentially of cross-linked HA. As used herein, the term “consist essentially of’ means that the composition includes the listed components andmay contain other components that do not materially affect the basic and novel characteristics of the invention. In some embodiments, the micronized cross-linked HA is devoid of non-cross- linked HA.

[0053] The micronized cross-linked HA has undergone a controlled miniaturization process to achieve a reduction in the average particle size of the macromolecule from which it originates, while preserving its inherent chemical structure, biological activity, and / or functional characteristics. The micronized cross-linked HA is further characterized by inherent stability or by stability achieved without reliance on a separate surface-stabilizing agent to maintain its structural integrity or properties in an acceptable carrier.

[0054] As used herein, the term “inherently stable” refers to cross-linked HA that maintains its structural integrity, physicochemical properties, and / or functional activity in an acceptable carrier without the need for an external or separate surface stabilizing agent. In other words, the cross-linked HA exhibits stability that is intrinsic to its own composition rather than being dependent on additional stabilizing materials or coatings.

[0055] In some embodiments, the molecule exhibits inherent stability for at least 3 days, at least 5 days, at least 10 days, or at least 30 days at 25°C, including any range of values in between. Each possibility represents a separate embodiment of the present invention. The inherent stability was examined by visible precipitation. As used herein, the term “Visible Precipitation” refers to the formation of a solid phase from a liquid medium, wherein the solid phase comprises insoluble or sparingly soluble particles present in a size and / or concentration sufficient to be detected by the unaided human eye under normal lighting conditions, without the use of magnification or analytical instrumentation.

[0056] As used herein, the term "retaining the key structural properties of the molecule it originates from" means that, despite the reduction in size, the downsized version preserves the primary structure, ensuring that any necessary folding or spatial organization critical to its activity is preserved. Additionally, the molecule retains its chemical functionality, meaning that active sites, binding domains, or reactive groups remain unaltered or only minimally modified. A person skilled in the art would appreciate that micronized cross-linked HA can be characterized by enhanced activity compared to the cross-linked HA it originates from. In some embodiments, retaining the key structural properties of the cross-linked HA is evidenced by a substantially similar FTIR profile before and after miniaturization

[0057] The reduction in particle size is calculated based on the following equation:100X (original cross-linked HA composition average particle size - micronized cross-linked HA composition average particle size) / (original cross-linked HA composition average particle size).For example, if the original cross-linked HA composition is characterized by an average particle size of 1500 nm, and the micronized cross-linked HA composition is characterized by an average particle size of 250 nm, then the reduction in particle size is 100X (1500nm - 250nm) / 1500nm = 83%.

[0058] In some embodiments, the micronized cross-linked HA composition is characterized by a reduction of between about 50% and 95%, between 50% and 90%, between 55% and 95%, between 65% and 95%, between 75% and 95%, or between 75% and 90%, in average particle size of the macromolecule it originates from, including any range or value in between. Each possibility represents a separate embodiment of the present invention.

[0059] A person skilled in the art would appreciate that typically there is a positive correlation between a particle's molecular weight and its average particle size; a higher molecular weight may lead to larger particles. Therefore, a reduction in particle size should correlate with a reduction in the particle's molecular weight. Other parameters that affect the relationship between particle size and molecular weight are cross-linking, aggregation, and molecular structure.

[0060] In some embodiments, the micronized cross-linked HA composition is characterized by a reduction of between about 30% and 95%, between about 50% and 95%, between 50% and 90%, between 55% and 95%, between 65% and 95%, between 75% and 95%, or between 75% and 90%, in molecular weight of the macromolecule which it originates from, including any range or value in between. Each possibility represents a separate embodiment of the present invention.

[0061] In some embodiments, the micronized cross-linked HA composition is characterized by a reduction of at least 30%, at least 40%, at least 45%, at least 50%, or at least 60%, in molecular weight of the macromolecule which it originates from, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0062] In some embodiments, the micronized cross-linked HA composition is characterized by an average particle size of between 105 nm and 500 nm, between 200 nm and 500 nm, between 300 nm and 500 nm, or between 300 nm and 450 nm, including any range or value in between. Each possibility represents a separate embodiment of the present invention.

[0063] As used herein, the term "average particle size" refers to an average diameter of a particle. The particle size is represented by DIO, D50, and D90, representing the average particle size of 10%, 50%, and 90% of the particles, respectively.

[0064] As used herein, the term "about" refers to a range of at least ±10%. For example, about 300 nm refers to values between 270 nm and 330 nm.

[0065] A person skilled in the art would appreciate that an average particle size can also be measured using scanning electron microscopy (SEM). Fig. 2 is an SEM image of a micronized crosslinked HA composition. An average particle size of between about 150 nm and 250 nm is seen, calculated using Image J.

[0066] In some embodiments, micronized cross-linked HA composition is characterized by a hygroscopy that is at least 1.5, at least 1.75, or at least 2 times greater than that of the crosslinked HA it originates from, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0067] As used herein, the term "hygroscopy" refers to a material's tendency to attract and hold water molecules from its surroundings. A person skilled in the art would appreciate that hygroscopy is a key property of HA, making it effective for hydration and moisture retention. Furthermore, the degree of hygroscopy can influence its performance in applications such as dermal fdlers and skincare. A higher hygroscopy level means better water retention and long-lasting hydration, while a lower level may indicate reduced swelling and a more controlled volumizing effect.

[0068] In some embodiments, the composition of the present invention further comprises an additive selected from the group consisting of a food supplement, antioxidants, fragrance, flavor, preservatives, colorants, emulsifiers, sweeteners, thickeners, stabilizers, acidulants, humectants, chelating agents, nutraceuticals, probiotics or prebiotics, including any combination thereof.

[0069] In some embodiments, the composition further comprises a pharmaceutical or a dermatologically acceptable carrier. In some embodiments, the composition is a pharmaceutical or a dermatological composition.

[0070] In some embodiments, the weight percentage of the pharmaceutical or the dermatologically acceptable carrier within the pharmaceutical or dermatological composition is between 0% and 99.99%, between 0% and 50%, between 5% and 99.99%, between 50% and 99.99%, between25% and 95%, or between 70% and 95%, including any value or range in between. Each possibility represents a different embodiment of the present invention.

[0071] As used herein, the term "carrier" refers to an inactive substance or formulation component used to deliver an active ingredient to the body in a safe, effective, and controlled manner. The carrier can increase stability, solubility, bioavailability, and controlled release of the active ingredient.

[0072] A pharmaceutical carrier is an inert substance used in drug formulations to help deliver an active ingredient safely and effectively. These carriers enhance the solubility, stability, and bioavailability of drugs, ensuring controlled release, better absorption, and targeting to specific areas in the body. Common pharmaceutical carriers include liposomes, polymers, and emulsions, which improve the delivery and effectiveness of the active drug and can also make the drug easier for patients to take (e.g., by masking taste or making it easier to swallow).

[0073] A dermatological carrier serves a similar role but in topical or transdermal applications. It helps deliver active ingredients to or through the skin, ensuring effective absorption while minimizing irritation. Dermatological carriers can improve the penetration of the active ingredient through the skin barrier, provide hydration, and allow for sustained release. Common carriers include creams, ointments, gels, emulsions, and liposomes, which ensure the active ingredients reach the intended layers of the skin for effective treatment with minimal side effects.

[0074] In some embodiments, the micronized cross-linked HA composition is an active ingredient in the pharmaceutical composition or dermatological composition. As used herein, the term "active ingredient" refers to the component in the composition that is responsible for the intended effect or therapeutic action. It is the substance that directly interacts with the body or target to produce the desired outcome, such as treating a disease, alleviating symptoms, or enhancing a certain function.

[0075] In some embodiments, the micronized cross-linked HA composition is an active ingredient in the pharmaceutical composition. Non-limiting examples of active ingredients include, but are not limited to, aloe vera, vitamins, anti-inflammatory agents, skin barrier repair agents, hydrating agents, antioxidants, moisturizers, anti-aging agents, firming agents, skin brightening agents, even-tone agents, acne actives, anti-inflammatory actives, soothers, UV protectors, or brightening agents, including any combination thereof.

[0076] In some embodiments, the micronized cross-linked HA composition is characterized by a cleaning degree of between 95% and 100%, between 95% and 99.99%, between 95% and 99%, or between 95% and 98%, including any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0077] In some embodiments, the pharmaceutical composition is for treatment or prevention of a disease, a disorder or a condition.

[0078] Non-limiting examples of a disease, a disorder, or a condition include, but are not limited to, joint disorders, wound healing, inflammation and autoimmune conditions, or skin and hair health, including any combination thereof.

[0079] In some embodiments, the pharmaceutical composition may be administered orally, parenterally, topically, via inhalation, transdermally, rectally, vaginally, or ophthalmically, including any combination thereof.

[0080] In some embodiments, the composition further comprises a cosmetically acceptable carrier. In some embodiments, the composition is a cosmetic composition.

[0081] In some embodiments, the weight percentage of the cosmetically acceptable carrier within the cosmetic composition is between 0% and 99.99%, between 0% and 50%, between 5% and 99.99%, between 50% and 99.99%, between 25% and 95%, or between 70% and 95%, including any value or range in between. Each possibility represents a different embodiment of the present invention.

[0082] In some embodiments, the micronized cross-linked HA composition is the sole active ingredient in the pharmaceutical composition.

[0083] In some embodiments, the cosmetic composition is for use in enhancing the appearance or feel of skin, hair, or nails, including any combination thereof. In some embodiments, enhancing appearance or feel is selected from the group consisting of improving fine lines and wrinkles, promoting skin rejuvenation, improving damaged hair, or strengthening nails, including any combination thereof.

[0084] In some embodiments, the cosmetic composition is topically administered, injectable, transdermally administered, or orally administered, including any combination thereof.

[0085] In some embodiments, the cosmetic composition is configured to penetrate the skin barrier. In some embodiments, the cosmetic composition penetrates the stratum comeum. In some embodiments, the cosmetic composition penetrates the epidermis. In some embodiments, thecosmetic composition penetrates the dermis. A person skilled in the art would appreciate that a composition that penetrates the dermis first has to penetrate the stratum comeum and then the epidermis. The deeper the active ingredients penetrate, the higher potential it has to affect deeper tissues.

[0086] The skin penetration of particulate formulations was evaluated using an iterative multi-phase optical property extraction technique (IMOPE). Briefly, light-intensity images of treated skin are acquired and processed by an iterative algorithm to reconstruct the phase of re-emitted light; the reconstructed phase distribution is compared to a theoretical light-propagation model to extract tissue scattering properties, including the reduced scattering coefficient (ps’). Because particles within tissue alter scattering, changes in the extracted scattering properties relative to baseline and / or control samples are used to detect the presence of particles at different depths and thereby estimate penetration depth and / or a penetration profile within the skin. A person skilled in the art would appreciate that there are several methods to evaluate the penetration of a molecule, including, for example, (i) Franz diffusion cell testing using excised human or animal skin to quantify permeation of an active into a receptor compartment, (ii) tape stripping to quantify material retained in the stratum comeum by sequential removal of comeocyte layers, (iii) confocal or multiphoton microscopy (optionally with fluorescently labeled particles) to visualize and quantify penetration depth in situ, (iv) Raman spectroscopy (e.g., confocal Raman) to generate depth-resolved chemical profiles within skin, and / or (v) histological sectioning followed by optical or electron microscopy to localize particles within skin layers.

[0087] A person skilled in the art would appreciate that the pharmaceutical composition, cosmetic composition, and dermatologic composition can be administered through various routes depending on the desired therapeutic effect, the nature of the drug, and patient-specific factors. Oral administration, including tablets, capsules, syrups, and suspensions, is the most common and convenient method, though it is subject to first-pass metabolism in the liver. Parenteral administration, which includes intravenous (IV), intramuscular (IM), subcutaneous (SC), and intradermal (ID) injections, allows for rapid or controlled drug release directly into the body. Topical and transdermal routes involve the application of creams, ointments, gels, and patches to the skin for localized or systemic effects. Inhalation methods, such as aerosols and inhalers, enable rapid absorption through the respiratory tract, often used for respiratory conditions. Sublingual and buccal administration, where drugs are placed under the tongue or against the cheek, provide quick absorption into the bloodstream without undergoing first-passmetabolism. Rectal and vaginal routes, using suppositories, creams, or foams, offer alternatives for localized or systemic treatment when oral administration is not feasible. Ophthalmic and otic administration involve the application of eye or ear drops for localized conditions, while intranasal delivery through sprays or drops can provide systemic or localized effects. Additionally, implantable devices, such as drug-releasing implants, allow for long-term controlled release of medications. Each method of administration has distinct advantages and limitations based on absorption rate, bioavailability, patient compliance, and potential side effects.

[0088] An exemplary injectable cosmetic composition comprises, by weight of composition, between 0.5% to 20% cross-linked HA, between 0.5% and 1.1% isotonicity agent, between 93% to 98% carrier, and between 0.5% to 4% anti-oxidant and moisturizer. In some embodiments, the carrier is or comprises phosphate-buffered saline, sodium phosphate buffer, or both. In some embodiments, the composition further comprises a pain relief agent.

[0089] According to another object of the present invention, there is provided a micronized crosslinked hyaluronic acid (HA) composition, comprising micronized cross-linked HA, a salt thereof, or a derivative thereof, including any combination thereof. The derivative thereof is selected from the group consisting of butanediol glycidyl ether cross-linked HA, divinyl sulfone cross-linked HA, polyethylene glycol diacrylate cross-linked HA, thiol-ene crosslinked HA, or carbodiimides cross-linked HA, including any combination thereof; wherein the micronized cross-linked HA composition (i) retains key structural properties of the cross-linked HA from which it originates; (ii) exhibits inherent stability or maintains its stability within the acceptable carrier without dependence on any separate surface-stabilizing agent. In some embodiments, the micronized cross-linked hyaluronic acid (HA) composition is characterized by an average particle size of between 105 run and 500 nm.

[0090] According to another object of the present invention, there is provided a pharmaceutical or a dermatologic composition comprising (a) the micronized cross-linked HA composition, a derivative thereof, or a salt thereof; and (b) a pharmaceutically or dermatologically acceptable carrier; wherein the micronized cross-linked HA composition retains the key structural properties of the cross-linked HA from which it originates.

[0091] In some embodiments, the pharmaceutical composition comprises between 0.1% to 25% micronized cross-linked HA, between 5% to 30% of at least one emollient, between 0.5% to 10% of at least one emulsifier, between 1% to 20% of at least one moisturizer, between 50%to 80% carrier, and between 0.1% to 10% of a polymer, by weight of the pharmaceutical composition. Each possibility represents a separate embodiment of the present invention.

[0092] In some embodiments, the amount of the micronized cross-linked HA is between 0.1% to 25%, between 0.1%to 20%, between 0.1%to 15%, between 0.1%to 10%, between 0.5%to 10%, or between 0.5% to 5%, by weight of the pharmaceutical composition, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0093] In some embodiments, the amount of at least one emollient is between 5% to 30%, between 5% to 25%, between 5% to 20%, between 5% to 15%, between 8% to 15%, or between 8% to 20%, by weight of the pharmaceutical composition, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0094] Non-limiting examples of at least one emollient include, but are not limited to, cetyl stearyl octanoate, C12-15 alkyl benzoate, caprylyl glycol, cetyl alcohol, isopropyl myristate, dimethicone, cocoa butter, glycereth-26, octyldecanol, butyrospermum parkii (shea) butter, dicaprylyl ether, tocopheryl acetate, glycereth-26, castor oil, castor isostearate succinate, di- ppg-3 myristyl ether adipate, ppg-3 benzyl ether myristate, ppg-3 benzyl ether myristate, myristyl lactate, silybum marianum ethyl ester, potassium cetyl phosphate, cetyl alcohol, stearyl alcohol, isoamyl laurate, ricinus communis (castor oil) seed oil, hydrogenated castor oil, beeswax, copemicia cerifera (carnauba) wax, triheptanoin, cl3-16 hydrogenated ethylhexyl olivate and hydrogenated olive oil unsaponifiables, coconut butter, grape seed oil, phytosteryl / behenyl / octyldodecyl lauroyl glutamate, hydrogenated polybutene, hydrogenated polydecene, glyceryl rosinate, C10-18 triglycerides, diisostearyl malate, helianthus annuus seed cera, acacia decurrens / jojoba / sunflower seed wax polyglyceryl-3 esters, helianthus annuus seed oil, isoamyl laurate, ethylhexyl stearate, carthamus tinctorius (safflower) seed oil, diisostearyl malate, polyphenylsilsesquioxane, caprylic / capric glycerides, caprylyl glycol, methicone, squalane, simmondsia chinensis (jojoba) seed oil, orbignya oleifera seed oil, lupinus albus seed oil, helianthus annuus (sunflower) seed oil, macadamia temifolia seed oil, olea europaea (olive) fruit oil, elaies guineensis (palm) oil, argania spinosa kernel oil, cocos nucifera (coconut) oil, safflower oil, solanum lycopersicum (tomato) seed oil, primus cerasus (bitter cherry) seed oil, alanites roxburghii seed oil, aleurites moluccanus seed oil, aryodendron orinocense seed oil, or persea gratissima (avocado) oil, including any combination thereof.

[0095] In some embodiments, the amount of at least one emulsifier is between 0.1% to 25%, between 0.1%to 20%, between 0.1%to 15%, between 0.1%to 10%, between 0.5%to 10%, or between0.5% to 5%, by weight of the pharmaceutical composition, including any value or range in between.

[0096] Non-limiting examples of at least one emulsifier include, but are not limited to, glyceryl stearate citrate, polyglyceryl-3 stearate, hydrogenated lecithin, olive oil, polyglyceryl-6 esters, sodium stearoyl lactylate, cetearyl alcohol, steareth-2, steareth-21, lecithin, sclerotium gum, glyceryl stearate, polyglyceryl-3 polyricinoleate, polyglyceryl-4 isostearate, sorbitan sesquioleate, cetearyl glucoside, myristyl alcohol, myristyl glucoside, C 14-22 alcohols, C12- 20 alkyl glucoside, arachidyl alcohol, behenyl alcohol, arachidyl glucoside, polysorbates, coco- glucoside, coconut alcohol, glyceryl stearate citrate, glyceryl oleate citrate, polyglyceryl-2 sesquioleate, PCA glyceryl oleate, lauryl glucoside, polyglyceryl-2 dipolyhydroxystearate, glycerin, sodium myreth sulfate, sodium C12-18 alkyl sulfate, sodium coco-sulfate, coco- betaine, PEG-40 hydrogenated castor oil, sodium cocoamphoacetate, hydrogenated vegetable oil, cocamidopropyl betaine, glyceryl oleate, dicocoyl pentaerythrityl distearyl citrate, sorbitan sesquioleate, cera alba, aluminum stearates, polyglyceryl-3 diisostearate, sodium cocoamphoacetate, glycerin, lauryl glucoside, sodium cocoyl glutamate, sodium lauryl glucose carboxylate, or polyquatemium, including any combination thereof.

[0097] A person skilled in the art would appreciate that when a range of carbon atoms is specified, it indicates that multiple hydrocarbon chain lengths can be used. For example, C12-C15 means that chains with 12, 13, 14, or 15 carbon atoms can be used.

[0098] In some embodiments, the amount of carrier is between 50% to 90%, between 55% to 90%, between 55% to 85%, between 50% to 85%, between 50% to 80%, or between 55% to 80%, by weight of the pharmaceutical composition, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0099] Non-limiting examples of carriers include, but are not limited to, water, glycerin, propylene glycol, butylene glycol, isopentyldiol, octyldodecanol, pentylene glycol, C12-15 alkyl benzoate, or polyol.

[0100] As used herein, the term "polyol" refers to any molecule with multiple hydroxy groups.

[0101] In some embodiments, the pharmaceutical composition further comprises an additive selected from the group consisting of fragrance, colorant, preservative, pH adjuster, surfactant, minerals, vitamins, humectants, skin conditioner, or flavor, including any combination thereof.

[0102] In some embodiments, the amount of preservative is between 0.1% to 2%, by weight of the pharmaceutical composition. In some embodiments, the amount of pH adjuster is within 0.1% to 5%, by weight of the pharmaceutical composition.

[0103] As used herein, the term "preservative" refers to a substance configured to prevent microbial growth, such as bacteria, fungi, and mold, and to extend shelflife by protecting the formulation from contamination and degradation.

[0104] According to another obj ect of the present invention, there is provided a cosmetic composition comprising (a) the micronized cross-linked hyaluronic acid composition, a derivative thereof, or a salt thereof; and (b) a cosmetically acceptable carrier; wherein the micronized cross-linked HA composition retains the key structural properties of the cross-linked HA it originates from.

[0105] In some embodiments, the cosmetic composition comprises between 0.1% to 25% micronized cross-linked HA, between 5% to 35% of at least one emollient, between 0.5% to 10% of at least one emulsifier, between 1% to 20% of at least one moisturizer, between 50% to 80% carrier, and between 0.2% to 2% of a thickening and stabilizing agent by weight of the cosmetic composition.

[0106] In some embodiments, the amount of the micronized cross-linked HA is between 0.1% to 25%, between 0.1%to 20%, between 0.1%to 15%, between 0.1%to 10%, between 0.5%to 10%, or between 0.5% to 5%, by weight of the cosmetic composition, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0107] In some embodiments, the amount of at least one emollient is between 5% to 30%, between 5% to 25%, between 5% to 20%, between 5% to 15%, between 8% to 15%, or between 8% to 20%, by weight of the cosmetic composition, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0108] Non-limiting examples of at least one emollient include but are not limited to cetyl stearyl octanoate, C12-15 alkyl benzoate, caprylyl glycol, cetyl alcohol, isopropyl myristate, dimethicone, cocoa butter, glycereth-26, octyldecanol, butyrospermum parkii (shea) butter, dicaprylyl ether, tocopheryl acetate, glycereth-26, castor oil, castor isostearate succinate, di- ppg-3 myristyl ether adipate, ppg-3 benzyl ether myristate, ppg-3 benzyl ether myristate, myristyl lactate, silybum marianum ethyl ester, potassium cetyl phosphate, cetyl alcohol, stearyl alcohol, isoamyl laurate, ricinus communis (castor oil) seed oil, hydrogenated castor oil, beeswax, copemicia cerifera (carnauba) wax, triheptanoin, cl3-16 hydrogenated ethylhexyl olivate and hydrogenated olive oil unsaponifiables, coconut butter, grape seed oil,phytosteryl / behenyl / octyldodecyl lauroyl glutamate, hydrogenated polybutene, hydrogenated polydecene, glyceryl rosinate, cl 0-18 triglycerides, diisostearyl malate, helianthus annuus seed cera, acacia decurrens / jojoba / sunflower seed wax polyglyceryl-3 esters, helianthus annuus seed oil, isoamyl laurate, ethylhexyl stearate, carthamus tinctorius (safflower) seed oil, diisostearyl malate, polyphenylsilsesquioxane, caprylic / capric glycerides, caprylyl glycol, methicone, squalane, simmondsia chinensis (jojoba) seed oil, orbignya oleifera seed oil, lupinus albus seed oil, helianthus annuus (sunflower) seed oil, macadamia temifolia seed oil, olea europaea (olive) fruit oil, elaies guineensis (palm) oil, argania spinosa kernel oil, cocos nucifera (coconut) oil, safflower oil, solanum lycopersicum (tomato) seed oil, primus cerasus (bitter cherry) seed oil, alanites roxburghii seed oil, aleurites moluccanus seed oil, aryodendron orinocense seed oil, or persea gratissima (avocado) oil, including any combination thereof.

[0109] In some embodiments, the amount of at least one emulsifier is between 0.1% to 25%, between 0.1%to 20%, between 0.1%to 15%, between 0.1%to 10%, between 0.5%to 10%, or between 0.5% to 5%, by weight of the cosmetic composition, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0110] Non-limiting examples of at least one emulsifier include but are not limited to glyceryl stearate citrate, polyglyceryl-3 stearate, hydrogenated lecithin, olive oil, polyglyceryl-6 esters, sodium stearoyl lactylate, cetearyl alcohol, steareth-2, steareth-21, lecithin, sclerotium gum, glyceryl stearate, polyglyceryl-3 polyricinoleate, polyglyceryl-4 isostearate, sorbitan sesquioleate, cetearyl glucoside, myristyl alcohol, myristyl glucoside, C14-22 alcohols, C12-20 alkyl glucoside, arachidyl alcohol, behenyl alcohol, arachidyl glucoside, polysorbates, coco- glucoside, coconut alcohol, glyceryl stearate citrate, glyceryl oleate citrate, polyglyceryl-2 sesquioleate, PCA glyceryl oleate, lauryl glucoside, polyglyceryl-2 dipolyhydroxystearate, glycerin, sodium myreth sulfate, sodium C12-18 alkyl sulfate, sodium coco-sulfate, coco- betaine, PEG-40 hydrogenated castor oil, sodium cocoamphoacetate, hydrogenated vegetable oil, cocamidopropyl betaine, glyceryl oleate, dicocoyl pentaerythrityl distearyl citrate, sorbitan sesquioleate, cera alba, aluminum stearates, polyglyceryl-3 diisostearate, sodium cocoamphoacetate, glycerin, lauryl glucoside, sodium cocoyl glutamate, sodium lauryl glucose carboxylate, or polyquatemium, including any combination thereof.

[0111] In some embodiments, the amount of carrier is between 50% to 90%, between 55% to 90%, between 55% to 85%, between 50% to 85%, between 50% to 80%, or between 55% to 80%, by weight of the pharmaceutical composition, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0112] Non-limiting examples of carriers include, but are not limited to, water, glycerin, propylene glycol, butylene glycol, isopentyldiol, octyldodecanol, pentylene glycol, C12-15 alkyl benzoate, or polyol.

[0113] In some embodiments, the amount of thickening and stabilizing agent is between 0.2% to 2%, between 0.2% to 1.8%, between 0.2% to 1.5%, between 0.4% to 2%, between 0.4% to 1.8%, or between 0.4% to 1.5%, by weight of the cosmetic composition, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0114] In some embodiments, the cosmetic composition further comprises an additive selected from the group consisting of fragrance, colorant, preservative, pH adjuster, surfactant, vitamins, minerals, humectants, pain relief, or flavor, including any combination thereof.

[0115] In some embodiments, the amount of preservative is between 0.1% to 1.5%, by weight of the cosmetic composition.

[0116] A person skilled in the art would appreciate that hyaluronic acid (HA), including linear and chemically cross-linked forms, is a naturally occurring glycosaminoglycan and a principal component of the extracellular matrix (ECM), where it can play structural and biological roles. HA can contribute to tissue hydration, viscoelasticity, and mechanical stability due at least in part to its high water-binding capacity and its ability to form hydrated matrices within connective tissues. In some contexts, high molecular weight HA is generally associated with tissue homeostasis and barrier-supportive or soothing activity, whereas lower molecular weight fragments may be associated with pro-inflammatory signaling responses. In addition to its structural role, HA has been reported to exhibit radical scavenging and protective properties in certain preclinical models, including reduction of oxidative stress-associated cellular damage and modulation of apoptosis-related pathways. Chemically cross-linked HA can exhibit increased resistance to enzymatic degradation and prolonged persistence, thereby providing sustained hydration, structural support, and mechanical scaffolding within treated tissues and / or on a treated surface. Collectively, these properties establish HA as a bioactive ECM component capable of influencing the tissue microenvironment, hydration status, mechanical properties, and cellular responses under physiological and stress-related conditions.

[0117] In some embodiments, the micronized cross-linked HA composition is configured to support one or more cosmetic and / or wellness-associated outcomes relating to skin appearance and / or skin conditioning, including improved hydration, improved barrier function, improved firmness or elasticity, improved smoothness, improved radiance, and / or reduced appearance offine lines. Without being bound by theory, such effects may be associated with the known biological and physicochemical activities of cross-linked HA, including formation of a hydrated matrix, increased water retention, reduced transepidermal water loss, and / or prolonged residence time on a treated tissue due to increased resistance to enzymatic degradation. Such outcomes may be evaluated using one or more measurable parameters, for example comeometry measurements, transepidermal water loss (TEWL), cutometry-derived elasticity and / or firmness parameters, and / or imaging-based assessments of wrinkle depth, roughness, and / or skin relief.

[0118] In some embodiments, the micronized cross-linked HA composition supports beneficial cellular responses, for example by promoting a hydrated extracellular microenvironment and / or by supporting normal cellular function and resilience under stress conditions. Without being bound by theory, such cellular benefits may include supporting one or more of: (i) cell viability in normal skin-associated cells and / or reduced markers associated with cellular stress; (ii) reduced oxidative stress burden; (iii) reduced markers associated with cellular senescence; and / or (iv) support of extracellular matrix homeostasis.

[0119] In some embodiments, the micronized cross-linked HA composition is configured to affect cellular longevity and / or cellular stress responses. Cellular longevity may be evaluated by determining one or more markers associated with cellular senescence, including but not limited to senescence-associated beta-galactosidase (SA-beta-gal) activity, pl6INK4a expression, p21 expression, and / or modulation of senescence-associated secretory phenotype (SASP) factors.

[0120] In some embodiments, the micronized cross-linked HA composition is configured to affect mitochondrial function and / or cellular energetics, mitochondrial function and / or cellular energetics may be assessed as optional exploratory endpoints, for example in cell culture or ex vivo skin models, by measuring mitochondrial membrane potential, oxygen consumption rate (OCR), mitochondrial DNA integrity, mitophagy-related markers, and / or reactive oxygen species (ROS) levels. Such mitochondrial endpoints may be used, for example, to characterize whether the composition supports mitochondrial integrity, and / or resilience to oxidative or environmental stress.

[0121] A person skilled in the art would appreciate that the miniaturization of a macromolecule, such as cross-linked hyaluronic acid, can be achieved through various methods, typically by breaking it down or modifying its structure to achieve a smaller size while retaining its essential properties and functions. This process can involve enzymatic degradation, where specificenzymes selectively cleave the macromolecule into smaller functional units. Chemical approaches, such as hydrolysis or oxidation, can also be employed to reduce molecular size. Furthermore, advanced nanotechnology techniques, including the self-assembly of smaller subunits or targeted chemical modifications, enable precise control over the macromolecule's dimensions. Computational modeling and molecular engineering strategies further facilitate the design of smaller, functionally similar analogs of the original macromolecule, optimizing them for applications in medicine, materials science, and biotechnology.

[0122] According to another aspect of the present invention, there is provided a method for treating a disorder, disease, or condition in a subject in need thereof, the method comprising contacting the subject's skin with a therapeutically effective amount of a dermatologic composition comprising (a) a micronized cross-linked hyaluronic acid (HA) composition, salt thereof, derivative thereof, or any combination thereof; and (b) a dermatologically acceptable carrier; wherein the micronized cross-linked HA composition retains the key structural properties of the cross-linked HA it originates from.

[0123] In some embodiments, the micronized cross-linked HA composition is characterized by an average particle size of between 105 nm and 500 nm, between 200 nm and 500 nm, between 300 nm and 500 nm, or between 300 nm and 450 nm, including any range or value in between. Each possibility represents a separate embodiment of the present invention.

[0124] In some embodiments, micronized cross-linked HA composition is characterized by a hygroscopy that is at least 1.5, at least 1.75, or at least 2 times greater than that of the crosslinked HA it originates from, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0125] As used herein, the term "subject" refers to an animal, more particularly to non-human mammals and human organisms. Non-human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses. Non-limiting examples of non-human animals include: horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, and pig. In one embodiment, the subject is a human. Human subjects may also include fetuses.

[0126] In some embodiments, the therapeutically effective amount is an amount sufficient to reduce, relieve, or both, at least one symptom of the disorder, disease, or condition.

[0127] As used herein, the term "therapeutically effective amount" refers to the amount of active ingredient required to reduce, prevent, or eliminate at least one symptom, or achieve a beneficial effect, including any combination thereof.

[0128] In some embodiments, the contacting is at least once a day for at least one consecutive day. A person skilled in the art would appreciate that the contacting may differ from subject to subject, where an authorized personnel such as a cosmetician or doctor determines the contacting time according to different parameters such as age, severity, gender, skin color, and type of disease, disorder, or condition.

[0129] In some embodiments, the disease is selected from the group consisting of rheumatoid arthritis, atopic eczema, psoriasis, atopic dermatitis, or any inflammation of the skin.

[0130] According to another aspect, there is provided a method for enhancing the appearance or feel of skin, hair, or nails, including any combination thereof, in a subject, the method comprising contacting the subject's skin with an effective amount of a cosmetic composition comprising (i) micronized cross-linked hyaluronic acid (HA) composition, a salt thereof, a derivative thereof, or any combination thereof; and (ii) a cosmetically acceptable carrier; wherein the micronized cross-linked HA composition retains key structural properties of the cross-linked HA from which it originates.

[0131] In some embodiments, the micronized cross-linked HA composition is characterized by an average particle size of between 105 nm and 500 nm, between 200 nm and 500 nm, between 300 nm and 500 nm, or between 300 nm and 450 nm, including any range or value in between. Each possibility represents a separate embodiment of the present invention.

[0132] In some embodiments, the micronized cross-linked HA composition is characterized by a reduction of between about 30% and 95%, between about 50% and 95%, between 50% and 90%, between 55% and 95%, between 65% and 95%, between 75% and 95%, or between 75% and 90%, in the molecular weight of the macromolecule from which it originates, including any range or value in between. Each possibility represents a separate embodiment of the present invention.

[0133] In some embodiments, micronized cross-linked HA composition is characterized by a hygroscopy that is at least 1.5, at least 1.75, or at least 2 times greater than that of the crosslinked HA from which it originates, including any value or range in between. Each possibility represents a separate embodiment of the present invention.

[0134] In some embodiments, the effective amount is an amount sufficient to enhance appearance or feel of skin, hair, or nails, including any combination thereof.

[0135] As used herein, the term "effective amount" refers to the amount of active ingredient required to reduce, prevent, or eliminate at least one symptom, or achieve a beneficial effect, including any combination thereof.

[0136] In some embodiments, enhancing the appearance or feel of skin, hair, or nails is selected from the group consisting of increased brightness, increased smoothness, increased plumpness, increased skin tone evenness, increased moisture, repair of hair and scalp damage, prevention of breakage, prevention of peeling, reduction of pigmentation, and reduction of wrinkles, including any combination thereof.

[0137] In some embodiments, the contacting is at least once a day for at least one consecutive day. A person skilled in the art would appreciate that the contacting may differ from subject to subject, where an authorized professional such as a cosmetician or doctor determines the contacting time according to different parameters such as age, severity, gender, skin color, and type of disease, disorder, or condition.EXAMPLES EXAMPLE 1 - Miniaturization Process

[0138] The preparation of cross-linked HA can be done either from HA or cross-linked HA. If HA is used, it should be cross-linked before miniaturization.

[0139] A person skilled in the art would appreciate that there are several crosslinking techniques available to crosslink HA, each offering unique advantages depending on the desired properties of the final product. Some of the commonly used methods include epoxide crosslinking, where reagents like 1,4-butanediol diglycidyl ether (BDDE) react with hydroxyl groups on HA to form stable ether bonds, providing enhanced durability and resistance to enzymatic degradation. Another widely used technique is carbodiimide crosslinking, which utilizes carbodiimide compounds such as l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to activate carboxyl groups on HA, facilitating the formation of amide bonds with available amine groups, resulting in biocompatible and biodegradable networks. Aldehyde crosslinking, using agents like glutaraldehyde, introduces covalent bonds through Schiff base formation between HA’s hydroxyl or amine groups and aldehyde functional groups, producing strong, tunable hydrogels. Additionally, urethane crosslinking involves the reaction of HA with isocyanate- containing crosslinkers to form urethane linkages, improving mechanical strength and waterresistance. Lastly, the Diels-Alder reaction, a reversible click chemistry approach, allows for thermally responsive crosslinking between furan and maleimide-modified HA, enabling self- healing properties and tunable degradation profdes. Each of these techniques is selected based on specific requirements such as mechanical stability, biocompatibility, and application in biomedical fields.Miniaturization Process:

[0140] A person skilled in the art would appreciate that there are several procedures for synthesizing micronized macromolecules. An exemplary synthesis comprises dissolving cross-linked HA composition in distilled water at a concentration of 1-5 mg / mL and adjusting the pH to 1-3. The solution is then heated to a temperature of between 60-90°C for a duration of between 1 to 6 hours. Once the desired level of cleavage is achieved, the reaction is neutralized by adding sodium hydroxide to restore the pH to neutral. The resulting micronized cross-linked HA are purified using dialysis or precipitation to remove residual acid and low-molecular-weight byproducts.

[0141] Other well-known methods for producing nanosized cross-linked HA include: (i) irradiating an HA aqueous solution with an electron beam; (ii) forming a water-in-oil (w / o) emulsion by mixing an oil phase containing a surfactant with a water phase containing HA and a water- soluble crosslinker dissolved in an aqueous basic solution, followed by crosslinking the HA within the w / o emulsion; and (iii) preparing a nanometer-scale cross-linked HA sodium gel based on CN1079984377A.

[0142] The composition was analyzed before and after the miniaturization process. Reference is now made to Fig. 1, a graph presenting Fourier Transform Infrared (FTIR) of cross-linked HA composition before (dashed line) and after (line) the miniaturization, according to some embodiments of the present invention. Fig. 1A demonstrates that the interactions of the composition before and after the miniaturization are similar. The peak at 3292 cm1represents stretching of aromatic carbon (=C-H), peaks at 1603 cm1and 1560 cm represent the stretching of a carboxyl (-C=O) of a carboxyl group and an acetamide, respectively, and the peak at 1151 cm1represents the stretching of -C-O. These results indicate that micronized cross-linked HA retains the key structural features of the cross-linked HA it originates from.

[0143] In addition, DLS analysis was conducted for a cross-linked HA composition (a) before miniaturization and (b) micronized cross-linked HA. Before miniaturization, the cross-linkedHA composition is characterized by an average particle size of about 600 nm, whereas after miniaturization the micronized cross-linked HA average particle size is about 250 nm.EXAMPLE 2 - Dermatologic Composition

[0144] A dermatologic composition was prepared according to some embodiments of the present invention. Formulation: 40% to 60% purified water, 0.5% to 10% humectant, 0.1% to 10% active ingredient, 0.1% to 10% moisturizer, 0.1% to 10% emulsifier, 3% to 28% emollient, 0.1% to 2% thickener, 0.1% to 2% preservative, and 0% to 0.5% fragrance.

[0145] In some embodiments, the active ingredient is or comprises an anti-inflammatory agent, skin barrier repair, Zinc PCA, micronized cross-linked HA, Vitamin B5, urea, and lactic acid.

[0146] A person skilled in the art would appreciate that a fragrance can be added in an amount ranging from 0.1% to 1%, by weight of the dermatologic composition. However, since this is a dermatologic composition and fragrances can be allergens, fragrance was not added to the following formulation.

[0147] Preparation of the dermatological composition involves heating the water and oil phases separately. Once both reach approximately 70°C, they are combined and homogenized to achieve a uniform consistency. As the mixture cools, active ingredients are incorporated, and the pH is adjusted to an ideal range of 5.0-6.0 if necessary.

[0148] The dermatologic composition, according to some embodiments of the present invention, was tested on a subject having atopic dermatitis. The subject applied the dermatologic composition twice a day for four days. Figs. 3A-3B show the subject's left foot before (3A) and after (3B) treatment.

[0149] Surprisingly, after just four days, a remarkable and noticeable reduction in the features of atopic dermatitis was observed. The subject reported clear improvement, describing a substantial sense of relief, significantly less irritation, and almost no itching after applying the cream. The difference was so pronounced that it was immediately felt, marking a major step toward skin comfort and healing. Additionally, a reduction in the depth of the cracks was observed, as indicated by the arrow.EXAMPLE 3 - Cosmetic Composition

[0150] A cosmetic composition was prepared according to some embodiments of the present invention. A cream was made according to some embodiments of the present invention, and the reactivity of micronized cross-linked HA was examined.

[0151] Formulation: 45% to 60% water, 5% to 20% emollient, 5% to 15% emulsifier, 0.1% to 10% active ingredient, 0.5% to 10% skin conditioner, 0.5% to 10% humectant, 0.2% to 1% preservative, 0.3% to 1% humectant, 0.1% to 1% thickening and stabilizing agent, 1% to 8% moisturizer, and 0%to 0.5% fragrance.

[0152] In some embodiments, the active ingredient is or comprises hydrating agents, moisturizers, anti-aging agents, firming agents, skin brightening agents, even-tone agents, acne actives, antiinflammatory actives, soothers, and UV protectors.

[0153] The cosmetic composition was prepared by heating the water and oil phases separately to 65- 70°C. Next, both phases were combined using high-shear mixing while the mixture was allowed to cool, with stirring continued throughout. Once the temperature dropped below 40°C, the active ingredients and preservatives were incorporated. Finally, the pH was adjusted to a range of 5.5 to 6.5, if necessary.

[0154] A subject applied the cream twice a day, morning and evening, for one week, and the effect of the cream on the fine lines and wrinkles on the forehead was examined. Fig. 4A demonstrates three deep wrinkles, while Fig. 4B shows that only one of the three deep wrinkles remains visible. A pronounced difference was observed after just one week of application.

[0155] As used herein, the term "VISIA" refers to an advanced skin imaging system used by skincare professionals to analyze and assess the skin's condition. It captures high-resolution images and provides detailed insights into various skin features such as wrinkles, pigmentation, pore size, and texture. The system also includes UV photography, revealing hidden sun damage. VISIA compares a person's skin to others of similar age and type and tracks the effectiveness of skincare treatments by monitoring changes over time. It is commonly used in dermatology and medical spas to create personalized skincare recommendations and treatments based on objective data.

[0156] In addition, seventeen participants with an average age of 52.9 applied the cosmetic cream made according to some embodiments of the present invention; and the difference in their skin age and wrinkles was examined using the VISIA. In 90% of participants, VISIA analysis presented a 15% percentile increase in wrinkle score, reflecting measurable improvement in skin smoothness and fine lines. As used herein, the term “percentile” refers to a comparative measure used by the VISIA Skin Analysis System. The VISIA system compares an individual’s skin characteristics to a large database of people of the same age, gender, and skin type. The percentile value indicates how the individual’s skin ranks within that group. For example, awrinkle score in the 80th percentile means the individual’s skin appears smoother and has fewer wrinkles than 80% of people in the same demographic group.

[0157] The average skin age of the participants reduced from 52.8 years to 49.9 years, indicating an overall improvement in measured skin condition and appearance. This reduction suggests that, on average, participants’ skin appeared approximately 2.9 years younger following treatment, as assessed by the VISIA Skin Analysis System.EXAMPLE 4 - Stability of Micronized Cross-Linked HA

[0158] A person skilled in the art would appreciate that Zeta potential measurements provide a quantitative indicator of particle stability. In general, particles with higher absolute zeta potential values, typically above ±20 to ±30 mV, exhibit stronger electrostatic repulsion and greater physical stability, whereas values near zero indicate a tendency toward aggregation or flocculation.

[0159] Representative data of different batches of cross-linked HA were used to prepare micronized cross-linked HA according to some embodiments of the present invention. The zeta potential of the cross-linked HA and micronized cross-linked HA was measured, and the data is summarized in Table 1.Table 1 : Zeta Potential of Micronized Cross-Linked HA and Cross-Linked HA from which it Originates

[0160] The observed increase in the magnitude of the zeta potential by a factor of at least 1.5 suggests the development of stronger electrostatic repulsion between particles, consistent with enhanced stability even in the absence of an external surfactant, furthermore, no visible phase separation was observed after storage under ambient conditions, supporting that the micronized crosslinked HA exhibits inherent stability within the acceptable carrier without reliance on any separate surface -stabilizing agent.EXAMPLE 5 - Antioxidant Capability of Micronized Cross-Linked HA

[0161] The antioxidant capability of micronized cross-linked HA was measured and compared to micronized HA. Fig. 5 is a graph presenting the antioxidant capability of each micronized molecule. It is clearly seen that the antioxidant capability of micronized cross-linked HA is about 5-fold that of micronized HA. These results demonstrate the potential of the cross-linked molecule to provide enhanced protection against oxidative stress, which may translate into improved efficacy in biomedical or cosmetic applications.

[0162] Antioxidant capability was measured using electron spin resonance (ESR), which is used in determining reactive oxygen species (ROS). Hydroxyl radicals were generated by performing a Fenton reaction (Fe2+ + H2O2 — > Fe3+ + OH* + OH-), and 5,5 -dimethyl- 1-pyrroline-N- oxide (DMPO) was added to bind the hydroxyl radicals and increase their half-life; the DMPO acts as a spin trap. The bound hydroxyl radicals can be easily detected using ESR.

[0163] The antioxidative properties of micronized CL-HA (m-CLHA) relative to those of CL-HA were evaluated by measuring the reduction of hydroxyl radicals generated in a Fenton reaction system. The reaction mixture (100 pL total volume) consisted of H2O2 (4400 pM, 10 pL), FeSCL (500 pM, 10 pL), MES buffer (pH 6.0, 65 pL), DMPO (5 pL), and either double-distilled water, CL-HA, or m-CLHA suspension (10 pL). The mixture was introduced into a capillary micropipette (BRAND GMBH, Germany) and placed in a standard rectangular Bruker ESR cavity (ER 4119 HS) of a Bruker ELEXSYS E500cw X-band ESR spectrometer. Spectra were recorded at room temperature under the following conditions: microwave frequency 9.8 GHz, microwave power 20 mW, sweep width 100 G (centered at 3518 G), and modulation amplitude 1 G.

[0164] The characteristic quartet signal of the DMPO-OH adduct was monitored, and the area under the ESR peaks was integrated to quantify the amount of hydroxyl radicals generated. A decrease in the signal intensity (or integrated area) in the presence of m-CLHA or CL-HA indicated hydroxyl radical scavenging activity. The relative scavenging activity (%) was calculated as: [(A_control - A_sample) / A_control] x 100, where A_control represents the integrated ESR signal area of the Fenton reaction without additive, and A_sample represents that obtained in the presence of m-CLHA or CL-HA. All measurements were performed in triplicate, and results are expressed as mean ± standard deviation.

[0165] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment.Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination were individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination were individually and explicitly disclosed herein.

[0166] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination.

[0167] The above illustrates and describes basic principles, main features, and advantages of the present invention. Those skilled in the art should appreciate that the above embodiments do not limit the present invention in any form. Technical solutions obtained by equivalent substitution or equivalent variations all fall within the scope of the present invention.

Claims

CLAIMSWhat is claimed is:

1. A micronized cross-linked hyaluronic acid (HA) composition, comprising micronized cross-linked HA, salt thereof, or derivative thereof, including any combination thereof;said derivative thereof is selected from the group consisting of butanediol glycidyl ether cross-linked HA, divinyl sulfone cross-linked HA, polyethylene glycol diacrylate cross-linked HA, thiol-ene cross-linked HA, or carbodiimides cross-linked HA, including any combination thereof;wherein said micronized cross-linked HA compositioni. is characterized by an average particle size of between 105 nm and 500 nm; ii. retains key structural properties of cross-linked HA it originates from; and iii. exhibits inherent stability or maintains its stability within said acceptable carrier without dependence on any separate surface-stabilizing agent.

2. The micronized cross-linked HA composition of claim 1 is characterized by a reduction of between about 50% and 95% in average particle size of macromolecule from which it originates.

3. The micronized cross-linked HA composition of claim 1 or 2, is characterized by a hygroscopy that is at least 1.5 times greater than that of said cross-linked HA it originates from.

4. The micronized cross-linked HA composition of any of claims 1 to 3, further comprises a pharmaceutical carrier.

5. The micronized cross-linked HA composition of claim 4, wherein said composition is a pharmaceutical composition.

6. The micronized cross-linked HA composition of claim 5, wherein said pharmaceutical composition is a dermatological composition.

7. The micronized cross-linked HA composition of claim 5 or 6, wherein in said pharmaceutical composition is for use in treatment or prevention of a disease, a disorder or a condition.

8. The micronized cross-linked HA composition of any of claims 1 to 3, further comprises a cosmetic acceptable carrier.

9. The micronized cross-linked HA composition of claim 8, wherein said composition is a cosmetic composition.

10. The micronized cross-linked HA composition of claim 9, wherein said cosmetic is for use enhancing appearance or feel of skin, hair, or nails, including any combination thereof.

11. The micronized cross-linked HA composition of claim 8 or 9, is characterized by at least penetration to a stratum comeum.

12. The micronized cross-linked HA composition of any of claims 1 or 11, is characterized by enhanced permeability compared to said cross-linked HA composition it originates from.

13. The micronized cross-linked HA composition of any of claims 1 to 11, further comprising an additive selected from the group consisting of food supplement, antioxidants fragrance, flavor, preservatives, colorants, emulsifiers, sweeteners, thickeners, stabilizers, acidulants, humectants, chelating agents, nutraceuticals, probiotics or prebiotics, including any combination thereof.

14. A pharmaceutical composition comprisinga. micronized cross-linked hyaluronic acid (HA), salt thereof, or derivative thereof including any combination thereof; andb. a pharmaceutical acceptable carrier;said derivative thereof is selected from the group consisting of butanediol glycidyl ether cross-linked HA, divinyl sulfone cross-linked HA, polyethylene glycol diacrylate cross-linked HA, thiol-ene cross-linked HA, or carbodiimides cross-linked HA, including any combination thereofwherein said micronized cross-linked HA compositioni. is characterized by an average particle size of between 105 nm and 500 nm; ii. retains key structural properties of cross-linked HA it originates from;iii. exhibits inherent stability or maintains its stability within said pharmaceutical acceptable carrier without dependence on any separate surface-stabilizing agent.

15. The pharmaceutical composition of claim 14, wherein said micronized cross-linked HA is characterized by a reduction of between about 50% and 95% in average particle size of macromolecule from which it originates.

16. The pharmaceutical composition of claim 14 or 15, wherein said micronized crosslinked HA is characterized by a hygroscopy that is at least 1.5 times greater than that of the cross-linked HA it originates from.

17. The pharmaceutical composition of any of claims 14 to 16, is for use in treatment of a disease, a disorder or condition.

18. The pharmaceutical composition of claim 17, wherein said disease, disorder or condition is selected from the group consisting of joint disorders, wound healing, inflammation and autoimmune conditions, or skin and hair health including any combination thereof.

19. The pharmaceutical composition of any of claims 14 to 18 is a dermatological composition.

20. A dermatologic composition comprisinga. micronized cross-linked hyaluronic acid (HA), salt thereof, or derivative thereof, including any combination thereof; andb. a dermatologic acceptable carrier;said derivative thereof is selected from the group consisting of butanediol glycidyl ether cross-linked HA, divinyl sulfone cross-linked HA, polyethylene glycol diacrylate cross-linked HA, thiol-ene cross-linked HA, or carbodiimides cross-linked HA, including any combination thereof;wherein said micronized cross-linked HA compositioni. is characterized by an average particle size of between 105 nm and 500 nm; ii. retains key structural properties of cross-linked HA it originates from;iii. exhibits inherent stability or maintains its stability within said pharmaceutical acceptable carrier without dependence on any separate surface-stabilizing agent.

21. A cosmetic composition comprisinga. micronized cross-linked hyaluronic acid (HA) salt thereof, or derivative thereof, including any combination thereof; andb. a cosmetic acceptable carrier;said derivative thereof is selected from the group consisting of butanediol glycidyl ether cross-linked HA, divinyl sulfone cross-linked HA, polyethylene glycol diacrylate cross-linked HA, thiol-ene cross-linked HA, or carbodiimides cross-linked HA, including any combination thereof;wherein said micronized cross-linked HA compositioni. is characterized by an average particle size of between 105 nm and 500 nm; ii. retains key structural properties of cross-linked HA it originates from;iii. exhibits inherent stability or maintains its stability within said pharmaceutical acceptable carrier without dependence on any separate surface-stabilizing agent.

22. The cosmetic composition of claim 21, wherein said micronized cross-linked HA is characterized by a reduction of between about 50% and 95% in average particle size of macromolecule from which it originates.

23. The cosmetic composition of claim 21 or 22, wherein said micronized cross-linked HA is characterized by a hygroscopy that is at least 1.5 times greater than that of the cross-linked HA it originates from.

24. The cosmetic composition of any of claims 21 to 23, is for enhancing appearance or feel of skin, hair, or nails, including any combination thereof.

25. The cosmetic composition of claim 24, wherein said enhancing appearance or feel is selected from the group consisting of improve fine line and wrinkles, promote skin rejuvenation, improve damaged hair, or strengthen nail, including any combination thereof.

26. The cosmetic composition of any of claims 21 to 25, is characterized by a penetration to the skin of at least a stratum comeum.

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