Methods for enhancing transdermal delivery of glycosaminoglycans (GAGs)

Abstract

A method for transdermal delivery of glycosaminoglycans (GAGs) is disclosed, which comprises applying ultrasound to the skin followed by topical administration of one or more GAGs, optionally complexed with a polysaccharide carrier. When the GAG ​​is hyaluronic acid complexed with modified starch, the transdermal delivery method facilitates delivery of high molecular weight hyaluronic acid to the deeper layers of the epidermis and dermis in a non-invasive, convenient, and painless manner. This transdermal delivery method can be applied to the treatment of skin aging phenomena associated with the depletion or loss of collagen and / or hyaluronic acid.

Description

Detailed Description of the Invention

[0001] FIELD OF THE INVENTION The present disclosure relates to methods for enhancing transdermal delivery of glycosaminoglycans (GAGs), and more particularly, but not exclusively, to ultrasound-enhanced transdermal delivery of hyaluronic acid (HA).

[0002] 〔background〕 Human skin aging is a complex biological process mediated by the combination of two independent factors. The first process is intrinsic or congenital aging, which is influenced by age-related hormonal changes, such as the decline in estrogen, androgen, and progesterone, associated with menopause and andropause. These hormone deficiencies lead to collagen degradation, dryness, loss of elasticity, skin atrophy, and wrinkles. The second process, extrinsic aging, is the result of exposure to external factors, primarily ultraviolet (UV) radiation. The key molecule responsible for improving skin moisture and collagen production is hyaluronan or hyaluronic acid (HA), a glycosaminoglycan (GAG; a long, unbranched polysaccharide composed of repeating disaccharide units) that forms the main component of the extracellular matrix (ECM). Young skin is hydrated due to the high amount of HA in the dermis. However, as we age, the amount of HA in the skin decreases, and by the time we reach adulthood, this amount is reduced to 5 percent of baseline. The combination of fibers and ECM provides skin with viscoelastic properties and resulting strength and resilience, but with age, disorganization and degradation of dermal fibers and HA occurs, resulting in a decrease in HA's ability to confer elasticity, density, and resistance to skin.

[0003] The stratum corneum (SC), the outer layer of skin, provides mechanical protection to the skin and acts as a barrier against water loss and the permeation of substances from the environment. In particular, the SC prevents the efficient penetration of large molecules (>500 Da). Therefore, the effective use of topically administered HA, which has a large molecular size, is limited due to skin permeability. In fact, topically applied HA does not fully penetrate the epidermis into the dermis. Therefore, when it is desired to prevent or treat the skin aging process, HA is usually delivered to deeper layers of the skin by injection. The major drawbacks of such invasive treatments can range from mild symptoms such as local pain or swelling to more serious problems such as severe damage due to penetration of the skin's blood vessels.

[0004] There is an unmet need for a non-invasive means for transdermal delivery of high molecular weight HA.

[0005] 〔summary〕 When applied topically to the skin, HA lacks the ability to penetrate the stratum corneum and remains on the surface of the skin, functioning as a skin surface moisturizer. Delivery of high-molecular-weight HA to deeper skin layers is highly desirable because it reaches more tissues and has a longer duration of action. Currently, transdermal delivery of high-molecular-weight HA for widespread application is achieved by intradermal (ID) injection of HA, i.e., injection delivered into the dermis.

[0006] The ability of applying low-frequency ultrasound to enhance biological membrane permeability, and in large part skin permeability, has been extensively studied by the present inventors, and a non-invasive delivery system for transdermal delivery of high molecular weight glycosaminoglycans (GAGs) such as HA has been envisioned, which may be utilized, among other things, in the treatment of skin aging. The present inventors have successfully performed the delivery of HA to the epidermis and dermis by applying ultrasound in combination with the use of chemically modified starch as an HA carrier.

[0007] Disclosed herein is a platform or system that combines ultrasound application with subsequent topical administration of HA (as well as other GAGs) complexed with a polysaccharide carrier. This platform provides a convenient, painless treatment for filling wrinkles, slowing the aging process, reducing aging indicators related to loss of mechanical properties, and restoring skin moisture for skin smoothing. The disclosed platform can further be utilized in multiple therapeutic procedures, including current HA injections, such as the treatment of knee pain caused by osteoarthritis.

[0008] In one aspect, the present disclosure provides a non-invasive method for preventing or treating a skin aging process in a subject in need thereof, comprising: (a) applying ultrasound treatment to a subject's skin surface for about 5 seconds to about 5 minutes; (b) topically administering to the sonicated skin surface at least one of free hyaluronic acid (HA) or HA complexed with a polysaccharide (HA-polysaccharide complex); and (c) optionally repeating at least one of steps (a) or (b) at least once; This relates to a method for non-invasively preventing or treating the skin aging process in a subject.

[0009] The disclosed method is suitable for transdermal delivery of HA of any molecular weight (MW), particularly for delivery of high molecular weight (HMW) HA (>1000 kDa).

[0010] The disclosed methods are useful for maintaining skin hydration, restoring or improving collagen production, slowing the aging process such as wrinkles, or reducing markers of aging related to skin atrophy or loss of mechanical properties such as loss of skin elasticity.

[0011] In another aspect, the present disclosure provides a method for transdermal delivery of one or more glycosaminoglycans (GAGs) in a subject in need thereof, comprising: (a) optionally forming a complex comprising one or more GAGs and at least one polysaccharide (GAG-polysaccharide complex); (b) applying ultrasonic treatment to the subject's skin surface for about 5 seconds to about 5 minutes; (c) topically administering one or more GAGs and / or one or more GAG-polysaccharide conjugates to the sonicated skin surface; (d) optionally applying an additional ultrasonic treatment to the skin surface for about 5 seconds to about 5 minutes; and (e) optionally, topically administering to the sonicated skin surface an additional amount of one or more GAGs and / or one or more GAG-polysaccharide conjugates; The present invention relates to a method for transdermally delivering one or more GAGs in a subject.

[0012] In some embodiments, step (a) is not applied.

[0013] In some embodiments, step (a) is applied and at least one GAG-polysaccharide conjugate is administered topically in step (c) and / or step (e).

[0014] In some embodiments, at least one of step (d) or step (e) is not applied. In some embodiments, at least one of step (d) or step (e) is applied once, twice, three times, or more times.

[0015] The GAG ​​delivered transdermally by the disclosed methods may be, for example, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or keratan sulfate, having a MW of 300 kDa to 8000 KDa, e.g., 500 kDa to 3000 kDa, or 300 kDa to 800 KDa.

[0016] The polysaccharides used in the contemplated methods may be at least one of starch, chitosan, pectin, cellulose, dextran, or galactan, and may optionally be chemically modified, for example, by substitution with one or more positively charged chemical moieties, such as, but not limited to, quaternary amine groups.

[0017] In some embodiments, starch substituted with quaternary amine groups (Q-starch) is utilized as a carrier for HA.

[0018] In a further aspect, the present invention provides a method for preparing starch comprising the steps of: + The present invention relates to a complex of hyaluronic acid and chemically modified starch, which is modified by substitution with one or more quaternary amine groups such as -.

[0019] In some embodiments, the disclosed complexes are characterized by a molar ratio of positively charged chemical moieties of the modified starch to negatively charged carboxyl groups of the hyaluronic acid (N / O molar ratio) that is from about 0.20 to about 3.00, e.g., from about 0.25 to about 1.5.

[0020] In yet another aspect, the present disclosure relates to a composition comprising a complex of hyaluronic acid and chemically modified starch as defined herein and at least one physiologically acceptable excipient.The intended composition can be a cosmetic composition or a therapeutic composition (i.e., a drug).

[0021] In yet another aspect, the present invention relates to a kit comprising: (a) at least one complex of hyaluronic acid and a chemically modified starch as defined herein, or a composition comprising same; (b) means for applying ultrasonic treatment; and (c) optionally, instructions and means for administering the complexed hyaluronic acid and / or composition to a subject.

[0022] Any of the complexes, compositions, and / or kits contemplated herein may be utilized to enhance non-invasive transdermal delivery of hyaluronic acid, preferably HMW HA, for purposes of, for example, anti-aging treatment.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS Some embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. Now, specific reference will be made to the drawings in detail, where it is emphasized that the particulars shown are shown by way of example and for purposes of illustrative discussion of the embodiments described herein. In this regard, the description using the drawings will make apparent to those skilled in the art how embodiments of the present disclosure may be practiced.

[0024] In the drawings: Figures 1A-1B show the size distributions of a complex of quaternary starch (Q-starch) with hyaluronic acid (HA) (Q-starch-HA complex) (1A) and free Q-starch and HA (1B) obtained using dynamic light scattering (DLS). The Q-starch-HA complex is characterized by an increased ratio between the positively charged amine groups (N) of Q-starch and the negatively charged carboxyl groups (O) of the HA backbone (N / O ratio); Figure 2 is a graph showing the size distribution (mean diameter) of free Q-starch, free HA, and Q-starch-HA complexes with N / O 0.25 measured using the NanoSight system; Figure 3 is a bar graph showing the average ζ potential (a function of particle surface charge) of free HA, Q-starch, and Q-starch-HA complexes characterized by increasing N / O ratios; Figures 4A-4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and freshly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C-4G); 5A-5B show the results of the application of Hylite™ Fluor 647 dye (HA) to the dermal layer of the dermal layer without ultrasound (US) pretreatment (5A) or after 5 minutes of US application (5B).Hylite Fluor 647 1A and 1B are bright-field confocal images of an exemplary pig ear skin cross-section after 24 hours of incubation with 0.3% (w / v) HA labeled with HA. The stratum corneum (SC), epidermal layer, and dermal layer are shown (bar: 20 μm). HA calculated for pixels in the exemplary rectangular cross-section shown by image j as a function of distance from the SC to a depth of 200 μm. Hylite Fluor 647 The fluorescence intensity is shown for each cross section. The vertical dashed lines represent the separation between layers. The horizontal dashed lines indicate the autofluorescence of the skin at the wavelength of the labeled HA; Figures 6A-6D show labeled Q-starch / HA complexes (Q-starch-HA) characterized by an N / O molar ratio of 0.25. Hylite Fluor 647 6A and 6C are confocal images of exemplary pig ear skin cross sections histologically stained after 24 hours of incubation with labeled Q-starch / HA complex (6A, 6B). Skin samples were either not pretreated with ultrasound prior to topical application of labeled Q-starch / HA complex (6A, 6B) or were treated with US for 5 minutes prior to complex application (6C, 6D). Figures 6A and 6C are confocal images showing intact nucleated cells in the skin layer below the stratum corneum (SC) (nuclear staining with 4',6-diamidino-2-phenylindole (DAPI)); Figures 6B and 6D are bright-field confocal images showing the SC, epidermal, and dermal layers (bar: 20 μm). Q-starch-HA Hylite Fluor 647 The fluorescence intensity of Q-starch-HA was calculated for pixels in the exemplary rectangular cross section shown by image j as a distance from the SC to a depth of 350 µm. Hylite Fluor 647 The fluorescence intensity is shown for each cross section. The vertical dashed lines represent the separation between layers. The horizontal dashed lines indicate the autofluorescence of the skin at the wavelength of the labeled HA; Figure 7 shows the amount of labeled HA (HA) in three layers of pig ear skin: SC (0-20 μm), epidermis (20-100 μm), and dermis (100-2000 μm). Hylite Fluor 647 ) Three groups of skin samples were observed: (i) labeled Q-starch-HA complex (Q-starch-HA) Hylite Fluor 647(ii) skin samples treated with ultrasound for 5 minutes and then treated with Q-starch-HA Hylite Fluor 647 (iii) a control group—skin samples treated with neither ultrasound nor the labeled complex for 24 hours; this group served for autofluorescence measurements. Fluorescence intensity was calculated by Imagej based on data recorded from confocal scans (three replicates ± SEM); and 8A-8D are confocal images of exemplary pig ear skin cross sections histologically stained after 24 hours of incubation with labeled Q-starch / HA with an N / O molar ratio of 0.25, where the Q-starch was labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch). 5-DTAF ), which appears as bright green staining in Images 8A and 8C, and HA is stained with Hylite™ Fluor 647 (HA Hylite Fluor 647 ) and appears as red staining in images 8B and 8D. The nuclei of intact cells beneath the SC are stained blue (DAPI stain). Skin samples were stained with labeled complex Q-starch. 5-DTAF -HA Hylite Fluor 647 The mice were not pretreated with ultrasound before topical administration of (8A, 8B) or were treated with US for 5 minutes before application of the complex (8C, 8D). Bar: 20 μm.

[0025] Detailed Description The present invention relates to a non-invasive means for enhancing the transdermal delivery of glycosaminoglycans (GAGs), and more particularly, but not exclusively, to the application of ultrasound to enhance the transdermal delivery of hyaluronic acid (HA).

[0026] In the context of the present disclosure, the term "transdermal delivery" should be broadly interpreted to include both (i) administration means that deliver a substance through the skin, i.e., onto the skin (topical), for example, by application of a solution, ointment, patch, etc. to promote its absorption systemically; and (ii) delivery to deeper skin layers, such as the epidermis and dermis, for example, through the upper outer stratum corneum (SC) skin layer to a depth of at least 350 μm below the SC. This latter mode of delivery is also referred to herein as "intradermal delivery" or "intradermal delivery." Thus, in any one of the embodiments described herein, transdermal delivery can apply to systemic delivery of GAGs through the skin and / or delivery of GAGs between skin layers.

[0027] The present disclosure is based on the inventors' discovery that applying ultrasound treatment to the skin can enhance the skin permeability of HA. The present disclosure is further based on the inventors' discovery that when HA is allowed to self-assemble with positively charged starch, i.e., starch substituted with a positively charged moiety such as a quaternary ammonium group, an HA-starch complex is formed that can be easily delivered transdermally after ultrasound application to the skin, and that it is stable in the deeper layers of skin tissue. Such a complex provides HA with higher tissue stability compared to free acidic glycosaminoglycans, and therefore provides HA with a longer retention time in the deeper layers of the skin, such as the epidermis and dermis.

[0028] The present inventors envision therapeutic and cosmetic hyaluronic acid-based treatment modalities in which HA is injected noninvasively percutaneously by utilizing ultrasound and the application of HA aggregates with quaternary starch (Q-starch). For example, the present inventors envision combining ultrasound application with Q-starch-HA complexes in skin aging treatment modalities, such as treating wrinkles, slowing the aging process, reducing aging indicators related to loss of mechanical properties, restoring skin moisture, and smoothing the skin. The combination of ultrasound application and HA complexation provides a convenient, noninvasive, and painless means for delivering HA to targeted skin layers, such as the dermis, further reducing the need for frequent transdermal administration of HA.

[0029] The examples disclosed herein demonstrate that ultrasound treatment applied to the skin before applying HA successfully inserted high molecular weight hyaluronic acid (1500 kDa) primarily into the upper skin layers. More significant penetration of HA into deeper layers of the skin (epidermis, dermis) occurred when HA was complexed with Q-starch before application to the skin. Complexes of Q-starch and HA (Q-starch-HA) were obtained at several molar ratios between the positively charged amine groups of Q-starch and the negatively charged carboxyl groups of HA (referred to herein as N / O molar ratios or simply N / O). The skin permeability experiments disclosed herein clearly demonstrate that ultrasound application successfully introduced larger amounts of Q-starch-HA complexes into deeper or lower layers of the skin and uniformly distributed these complexes therein.

[0030] The skin is the largest organ in the human body, measuring approximately 2m in a healthy adult. 2It has a surface area of ​​1000 m², is only a few millimeters thick, and accounts for approximately 15% of an adult's body weight. It is a heterogeneous, multilayered tissue that contains nearly one-third of the circulating blood. Skin is a barrier to physical and chemical penetration into the body from the environment and has several functions, including protection and resistance against environmental attacks, protection against infectious agents, protection from dehydration, and wound repair and regeneration. Two major tissue layers are traditionally recognized as constituting human skin: the outermost layer is the epidermis, and the second layer is the dermis.

[0031] The epidermis, approximately 0.07–1.4 mm thick, is composed primarily of cells called keratinocytes and is organized into five layers representing different stages of cellular life in the epidermis. The arrangement of layers from inner to outer is: (1) the basal layer (stratum basale), composed of vertically arranged columnar cells; (2) the spinous layer (stratum spinosum), composed of flattened polyhedral cells with short spines; (3) the granular layer (stratum granulosum), composed of flattened granular cells; and (4) the clear layer (stratum lucidum), composed of several layers of clear, transparent cells with indistinct or absent nuclei. In the epidermis of the general body surface, the stratum corneum (SC) is usually absent, and the stratum corneum (SC) is composed of flat, hexagonal, keratinized, non-nucleated cells called "keratinocytes," held together by lipids and desmosomes, commonly referred to as a brick-and-mortar structure. Desmosomes are specialized interkeratinocyte junctions formed by proteins, which, together with lipids, maintain the integrity of the SC. The keratinocytes in this outermost surface of the epidermis are dead and filled with keratin, forming a strong, hydrophobic (13% water) protective layer (also known as the keratin layer). Lipids form several bilayers surrounding the keratinocytes. The stratum corneum contains 15–20 layers of keratinocytes and, in its dry state, is 10–15 μm thick. Upon hydration, the stratum corneum swells significantly, its thickness can reach 40 μm, and its permeability increases.

[0032] The stratum corneum is the primary transport barrier for external substances. Furthermore, the SC prevents water loss from the skin surface. It is also involved in immunological and inflammatory processes. Given its barrier properties and water resistance, the stratum corneum is the primary layer that limits drug absorption through the skin.

[0033] Cells in the layers below the SC divide to replenish supplies. The epidermis is not vascularized, so living keratinocytes in the epidermal layer receive nutrients from the dermis, which is separated from the epidermis by a basement membrane (dermal-epidermal junction (DEJ)).

[0034] The dermis (true skin) is the fibrous inner layer of skin just below the epidermis. It is derived from the embryonic mesoderm and ranges in thickness from 0.05 cm to 0.3 cm. The dermis contains fibroblasts, histiocytes, and mast cells. Its composition is primarily fibrous, consisting of both collagen and elastic fibers produced by fibroblasts. Between the fibrous components is an amorphous extracellular "matrix" containing glycosaminoglycans such as hyaluronic acid, proteoglycans, and glycoproteins. The dermis provides structure, elasticity, flexibility, and strength to the skin, protecting it from mechanical injury. The dermis helps maintain epidermal properties and repair and restore the skin after injury.

[0035] The dermis is composed of two zones: a superficial thin layer that interdigitates with the epidermis (papillary layer, or papillary dermis), and a deeper coarse reticular layer (or reticular dermis). The papillary dermis is richly supplied with blood and lymphatic vessels, as well as nerves and nerve endings. The reticular dermis is in contact with the hypodermis (innermost layer of skin) and is composed of thick collagen fibers that provide strength and elasticity to the skin and contains hair follicles, sweat glands, and sebaceous glands.

[0036] Types I and II collagen make up approximately 75% of the dry weight of the dermis.

[0037] The primary route of skin permeation is through the intact epidermis, and two main pathways have been identified: the intercellular pathway through the lipids of the stratum corneum and the transcellular pathway through corneocytes. In both cases, drugs must diffuse into the intercellular lipid matrix, which is recognized as the primary determinant of drug absorption through the skin. Drug transport in the skin can be viewed as a process involving several steps: (a) drug dissolution and release from the formulation; (b) drug partitioning into the stratum corneum; (c) drug diffusion across the stratum corneum, primarily via intercellular lipids; (d) drug partitioning from the stratum corneum to the viable epidermal layers; (e) diffusion across the viable epidermal layers into the dermis; and (f) drug absorption by capillaries that reach the systemic circulation. The SC, a barrier to water loss from the environment and penetration of substances, provides mechanical protection to the skin, preventing efficient penetration of large molecules (>500 kDa). Therefore, the primary transdermal delivery route for large molecules is often invasive, e.g., via injection.

[0038] Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are negatively charged polysaccharide compounds containing amino sugars or monosaccharides in which the -OH group is replaced by an NH group, such as D-glucosamine and D-galactosamine. They are composed of repeating disaccharide units, and their functions in the body are widely known and determined by their molecular structure. For example, GAGs play important roles in cell signaling and a vast number of biochemical processes. Some of these processes include regulating cell growth and proliferation, promoting cell adhesion, anticoagulation, and wound repair. Four major groups of GAGs are classified based on their core disaccharide unit: heparin / heparan sulfate, chondroitin sulfate / dermatan sulfate, keratan sulfate, and hyaluronic acid. The main categories of GAGs differ based on the type of monosaccharide and whether or not they are modified by sulfation.

[0039] In one aspect, the present disclosure provides a non-invasive method for transdermal delivery of one or more glycosaminoglycans (GAGs) in a subject in need thereof, comprising: (a) applying ultrasonic treatment to the skin surface of the subject for about 5 seconds to about 5 minutes; (b) topically administering one or more GAGs to the sonicated skin surface; (c) optionally applying an additional ultrasonic treatment to the skin surface for about 5 seconds to about 5 minutes; and (d) optionally, topically administering to the sonicated skin surface an additional amount of one or more GAGs; This relates to a method for non-invasive transdermal delivery of GAGs.

[0040] Typically, but not necessarily, GAGs delivered transdermally upon ultrasound pre-application have a molecular weight <500 kDa. Higher molecular weight GAGs, e.g., GAGs with a molecular weight ranging from 500 kDa to 8000 kDa, can be delivered in accordance with the present disclosure by assembly or complexation with certain carrier polymers, including, but not limited to, polysaccharides such as starch, chitosan, pectin, cellulose, dextran, or galactans, optionally functionalized polysaccharides.

[0041] Such complexes have previously been utilized by the present inventors as non-viral carriers of microRNA (miRNA) for the treatment of psoriasis, small interfering RNA (siRNA) for the treatment of ovarian cancer, and PI3P for the treatment of liver insulin resistance (see, for example, Amar-Lewis et al., Journal of Controlled Release 185:109-120, 2014; Lifshiz Zimon et al., Journal of Controlled Release 284:103-111, 2018). The use of polysaccharides as delivery vectors is considered advantageous due to their natural characteristics, such as biodegradability, biocompatibility, low immunogenicity, and minimal cytotoxicity. In particular, starch can be carefully designed and characterized in terms of molecular weight and modification to address safety and efficiency issues in delivery.

[0042] In some embodiments, the carrier polysaccharide utilized in contemplated methods is a functionalized starch.

[0043] Starch has the general formula (CH 10 O5) n Starch is a group of polysaccharides that is the primary storage form of carbohydrates in plants and one of nature's energy reserves. Starch is a biodegradable polymer composed of D-glucose residues, consisting of 20% amylose and 80% amylopectin. Amylose contains α-1,4 bonds, while amylopectin also contains α-1,6 bonds. It is found primarily in the pith of plant seeds, fruits, tubers, roots, and stems, particularly in corn, potato, wheat, and rice. While its appearance varies greatly depending on the source, it is generally prepared as a white, amorphous, tasteless powder. Starch has many favorable characteristics, including low toxicity, biocompatibility, stability, low cost, hydrophilicity, and availability of reactive sites for chemical modification.

[0044] For starch to be an effective carrier of GAGs, starch, being an electrically neutral polysaccharide, must undergo specific modification (also referred to herein as "functionalization"), e.g., by attaching positively charged groups to the starch. The term "quaternized starch" (Q-starch) refers to a starch molecule having a backbone that has undergone specific modification, such as the substitution or addition of at least one quaternary moiety or group. A quaternary moiety or group is defined herein as a cation consisting of a central positively charged atom bearing four substituents. Such cations are also referred to herein as "quaternary cations." A "quaternary compound," as defined herein, is a compound that is or has a quaternary cation. The most well-known quaternary compounds are quaternary ammonium salts (N) with a central positively charged nitrogen atom. + R4; R is a substituent). Other examples include substituted phosphonium salts (R4P + ), and substituted arsonium salts such as arsenobetaine (R4As +For example, quaternized potato starch can be obtained by substitution with quaternary groups, which gives Q-starch cationic properties. Q-starch can bind to molecules with negatively charged groups by self-assembly of complexes.

[0045] In the context of the embodiments described herein, Q-starch refers primarily to starch that has been quaternized by substitution with one or more quaternary amine moieties.

[0046] In a further aspect, the present disclosure provides a non-invasive method for facilitating transdermal penetration of one or more GAGs having a molecular weight of 500 kDa to 8000 kDa into deeper layers of the skin, i.e., through the stratum corneum (SC) into deeper layers of the epidermis and, for example, into the dermis, comprising: (a) applying ultrasound treatment to a subject's skin surface for about 5 seconds to about 5 minutes; (b) topically administering to the sonicated skin surface one or more GAGs complexed with at least one polysaccharide; (c) optionally applying an additional ultrasonic treatment to the skin surface for about 5 seconds to about 5 minutes; and (d) optionally, topically administering an additional amount of GAG-polysaccharide complex to the sonicated skin surface, thereby promoting penetration of the GAG ​​into deeper layers of the skin.

[0047] Steps (c) and (d) may be repeated one, two, three or more times as needed.

[0048] The "deep layer of the skin" referred to herein refers to a layer below the stratum corneum, such as the inner epidermis layer, such as the stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale, and / or the dermis layer. The "deep layer of the skin" also refers to a depth of 0 to about 2000 μm below the stratum corneum, such as about 0 to about 10 μm, about 5 μm to about 20 μm, about 10 μm to about 30 μm, about 20 μm to about 40 μm, about 30 μm to about 60 μm, about 50 μm to about 80 μm, about 60 μm to about 100 μm, about 80 μm to about 120 μm, about 100 μm to about 150 μm, about 140 μm to about 200 μm, about 180 μm to about 250 μm, about 200 μm to about 30 ...0 μm to about 600 μm, about 50 μm to about 80 μm, about 60 μm to about 100 μm, about 80 μm to about 120 μm, about 100 μm to about 150 μm, about 140 μm to about 200 μm, This refers to about 270 μm, about 250 μm to about 300 μm, about 280 μm to about 350 μm, about 320 μm to about 400 μm, about 250 μm to about 500 μm, about 450 μm to about 600 μm, about 500 μm to about 800 μm, about 650 μm to about 900 μm, about 800 μm to about 1000 μm, about 900 μm to about 1500 μm, or about 1000 μm to about 1800 μm, as well as any subranges and individual depths therebetween.

[0049] Glycosaminoglycans that can be delivered transdermally using the contemplated methods described herein include, but are not limited to, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. In some embodiments, any of these GAGs has a molecular weight of 500 kDa to 5000 kDa.

[0050] Heparan sulfate is known as a pharmacological target for cancer therapy. Notable functions of heparan sulfate include organizing the extracellular matrix (ECM) and regulating cell growth factor signaling by acting as a bridge between receptors and ligands. In the extracellular matrix, heparan sulfate interacts with many compounds, including collagen, laminin, and fibronectin, to promote cell-to-cell and cell-to-extracellular matrix adhesion. In malignant tumors such as melanoma, degradation of heparan sulfate in the extracellular matrix by the action of the enzyme heparanase leads to malignant cell migration and metastasis. This mechanism makes heparanase and heparan sulfate viable pharmacological targets for preventing cancer metastasis.

[0051] Heparin is used as an anticoagulant by a mechanism involving its interaction with the protein antithrombin III (ATIII), resulting in a conformational change in ATIII that enhances its ability to function as a serine protease inhibitor of coagulation factors. Different molecular weights of heparin have been shown to exhibit varying clinical anticoagulant potency.

[0052] Chondroitin sulfate is known for its clinical use as a disease-modifying osteoarthritis drug (DMOAD), particularly for symptomatic pain relief and structural modification in osteoarthritis (OA). The pain-relieving properties of chondroitin sulfate in OA are related to its anti-inflammatory properties. One of the major pathophysiological causes of OA is related to the loss of chondroitin sulfate from articular cartilage, leading to inflammation and catabolism of cartilage and subchondral bone. The role of chondroitin sulfate structural modification in OA is attributed to its role in stimulating type II collagen and proteoglycan (PG) production in both articular cartilage and synovium. This anabolic effect of chondroitin sulfate prevents further tissue damage and remodeling of synovial tissue.

[0053] Keratan sulfate has functional roles in both the cornea and the nervous system. The cornea is the most abundant known source of keratan sulfate in the body, followed by brain tissue. Keratan sulfate's role in the cornea includes regulating the spacing of collagen fibrils, which is essential for optical transparency, and optimizing corneal hydration during development through its interaction with water molecules. As with other GAGs, the degree of sulfation of keratan sulfate determines its functional state. Keratan sulfate has also been shown to play an important regulatory role in neural tissue development. Various subgroups of keratan sulfate in the brain play important roles in stimulating microglial cell proliferation and promoting axonal repair after injury.

[0054] Hyaluronic acid (HA) or hyaluronan has the simplest structure of all GAGs. It is a long, homogeneous, unbranched polysaccharide consisting of two repeating disaccharide units: D-glucuronic acid and N-acetyl-D-glucosamine, linked together via alternating β-1,4 and β-1,3 glycosidic bonds. The number of repeating disaccharide units in an HA molecule can reach more than 10,000 units in the human body. Hyaluronic acid is a major component of the extracellular matrix and is most abundant in the skin (approximately 50% of total HA is present in the skin, both in the dermis and epidermis), accounting for 15% of total body weight. Hyaluronic acid is present in all tissues and fluids of the body, including the vitreous body, joints, cornea, umbilical cord, and synovial fluid. Hyaluronic acid plays an important role in the synthesis of extracellular matrix molecules and epidermal cell interaction with the surrounding environment. It regulates cellular immunity by preventing infection and preventing allergic phenomena.

[0055] Hyaluronan production is controlled by fibroblasts, keratinocytes, or chondrocytes. In tissues such as skin and cartilage, where HA constitutes the majority of the tissue mass, HA synthesis levels are very high. Hyaluronan has a dynamic turnover rate, with a half-life of 3–5 minutes in blood, less than 1 day in skin, and 1–3 weeks in cartilage. It degrades into fragments of various sizes either enzymatically by hyaluronidase or non-enzymatically by a free radical mechanism in the presence of reducing agents such as ascorbic acid, thiols, ferrous, or cuprous ions, a process that requires the presence of molecular oxygen.

[0056] Hyaluronic acid is best known for its ability to attract water molecules. In physiological solutions, the carboxyl groups of HA are negatively charged (anionic), and HA can form salts with mobile cations. These salts are highly hydrophilic and, as a result, are surrounded by water molecules. HA's highly polar structure allows it to bind 10,000 times its own weight in water. Water molecules bind to the HA carboxyl and acetamido groups through H-bonds, stabilizing the secondary structure of the biopolymer, which is described as a single-stranded left-handed helix (double helix) with two disaccharide residues per turn. In aqueous solution, these double helices form a double-stranded, β-sheet tertiary structure due to hydrophobic interactions and intermolecular H-bonds, which allows the polymer chains to aggregate, forming an extended network. The HA network strengthens with increasing molecular weight (MW) and concentration. These properties allow HA to play an important role in the lubrication of synovial joints and the wound healing process.

[0057] Hyaluronic acid has a fairly wide molecular size / weight range (10 5 ~10 7 Da) and occurs in a vast number of configurations and shapes, depending on its size, salt concentration, pH, and associated cations. The biological function of HA is highly dependent on its size: high molecular weight hyaluronan (HMW HA) chains (>5 × 10 5Da) have space-filling, anti-angiogenic, immunosuppressive, cell growth inhibitory, and endothelial cell migration properties and are commonly applied in the pharmaceutical field for various applications (e.g., cancer treatment, osteoarthritis treatment, ophthalmic surgery, plastic surgery, drug delivery, and wound healing); 4 ~10 5 Medium-sized hyaluronan chains (between 100 kDa and 100 kDa) are involved in ovulation, embryogenesis, and wound repair; 3 ~2×10 4 Low molecular weight HA (LMW HA) chains (between 100 and 150 Da) are proinflammatory, immunostimulatory, and angiogenic, whereas small HA oligomers (400-4000 Da) are antiapoptotic and inducers of heat shock proteins. Low molecular weight HA and smaller oligosaccharides can be produced naturally in the body or artificially by controlled depolymerization of HMW HA using physical treatments (heat treatment, pressure), irradiation, ultrasound application, acid treatment, radical oxidation, and enzymatic hydrolysis with hyaluronidase.

[0058] Hyaluronic acid is commonly utilized in the cosmetics and food industries and is used exogenously by clinicians to promote tissue regeneration and skin repair. It has demonstrated safety and efficacy for these purposes and has been approved by the Food and Drug Administration (FDA) as a transdermal filler. Some HA-based products are already on the market and / or in established clinical practice, while others are currently undergoing research to confirm their effectiveness.

[0059] In cosmetics, HA has shown promising efficacy in promoting skin tone, elasticity, and improving aesthetic scores. For example, its use in cosmetic formulations as a moisturizing active ingredient to restore a physiological microenvironment typical of young skin is well known and widely used. For cosmetic applications, HA is classified according to its molecular weight. Hyaluronic acid of 20–300 kDa can penetrate the stratum corneum, while 5 kDa HA penetrates deeper into the epidermis. Meanwhile, HA of higher molecular weights (500–1500 kDa) remains normally on the skin's surface and is unable to penetrate the stratum corneum (SC).

[0060] Hyaluronic acid utilized in the embodiments described herein encompasses any form of commercially available or custom-made HA produced by any of the techniques known in the art, such as, but not limited to, extraction from animal sources or microbial fermentation (e.g., fermentation of strains of the bacteria Streptococci).

[0061] Some embodiments relate to the use of chemically modified HA. Chemical modifications of HA primarily involve two functional sites: hydroxyl (probably the primary alcohol functional group of N-acetyl-D-glucosamine) and carboxyl. These functional groups can be modified by two techniques that are based on the same chemical reaction but result in different products: conjugation and crosslinking. Conjugation consists of grafting one or more monofunctional molecules onto the HA chain, each forming a single covalent bond, while crosslinking uses a multifunctional compound to link different chains of native or conjugated HA together through two or more covalent bonds. Crosslinked hyaluronan can be prepared from native HA (direct crosslinking) and / or HA conjugates (i.e., HA covalently bound to one or more functional groups). In direct crosslinking of native HA molecules, the hydroxyl and carboxyl groups can be crosslinked via ether and ester bonds, respectively. In some embodiments, HA is chemically modified prior to its crosslinking to introduce other chemically reactive groups. For example, HA may be treated with acid or base to undergo at least partial deacetylation, resulting in the presence of free amino groups, which can then be crosslinked via amide (-C(O)-NH-); imino (-N=CH-) or secondary amine (-NH-CH-) bonds. The imino bonds can be converted to amine bonds in the presence of a reducing agent.

[0062] Conjugation and crosslinking are commonly performed for a variety of purposes. For example, conjugation provides crosslinking with various molecules to obtain carrier systems with improved drug delivery properties or prodrugs. Crosslinking can further improve the mechanical, rheological, and swelling properties of HA, reduce its degradation rate, and provide HA derivatives with longer residence times at the application site and greater release characteristics. The higher the degree of crosslinking, the lower the water absorption capacity of the crosslinked HA and the greater its stability in aqueous solutions. Furthermore, doubly crosslinked HA exhibits greater stability against degradation by hyaluronidase and free radicals, resulting in increased biological stability. For example, crosslinked HA has been used in cosmetic applications in the field of skin aging.

[0063] The term "hyaluronic acid," as used herein, encompasses native HA of any molecular weight known in the art, as well as HA derivatives, including any chemically or physically modified HA, such as, but not limited to, HA conjugates and cross-linked HA.

[0064] Hyaluronan homeostasis changes with age and due to external and internal processes and agents, such as sun exposure, that cause HMW HA degradation. The HA content of the dermis is significantly higher than that of the epidermis. Both epidermal and dermal cells are capable of synthesizing HA throughout our lives. However, skin cells lose their ability to produce optimal amounts of HA during the aging process. The primary histochemical change observed in aging skin is a significant decrease in epidermal HA, while HA remains present in the dermis. Thus, the epidermis loses key molecules involved in binding and retaining water molecules, resulting in a loss of skin moisture. In the dermis, the primary age-related change is an increase in HA's binding strength (functional affinity) with tissue structures, accompanied by a concomitant loss of HA extractability. This parallels the gradual cross-linking of collagen and the steady loss of collagen extractability with age. The decrease in HA production is also accompanied by the loss of suppleness, elasticity, and skin tone that characterize aging skin.

[0065] To maintain the aesthetic appearance of the skin and treat the signs of "dermatological" aging, it is recommended to continue "topping up" the skin with HA from puberty onwards. Today, the treatments available for adding HA to the skin are serums, injections, and oral ingestion. It is already known that orally ingested HA does not show any benefit to the appearance of the skin, since skin cells cannot extract HA from the bloodstream. HMW natural HA (>6 x 10 5 Topical application of HMW HA (HMW HA) is challenging, primarily because its large size prevents efficient penetration into deeper skin layers. Instead, it forms a film that acts as a barrier against water loss. Furthermore, due to its favorable gelling properties, topical application of HA leads to a hydrating effect in the uppermost layers of the skin; the accumulating water can swell and open the dense structure of the stratum corneum, resulting in increased HA penetration into the upper layers of the epidermis. Thus, HMW HA has a beneficial effect on hydration of the upper epidermal layers, which is manifested by subcutaneous transepidermal water loss. The hydration capacity of the skin depends on the molecular size of HA, and for this reason, HMW HA (approximately 1 MDa) is commonly added to cosmetic formulations.

[0066] However, the penetration of HA, especially HMW HH, into deeper layers of the skin is still very slow. The penetration properties (e.g., anti-aging effects) of topically applied HMW HA can be improved by combining HA with a skin-penetrating carrier. In most cases, and in all cases of HA application to deeper skin layers, HA, and especially cross-linked HA, is injected in a rather painful application procedure that can sometimes cause inflammatory complications and bacterial infections.

[0067] In a further aspect, the present disclosure relates to a non-invasive method for facilitating transdermal delivery of hyaluronic acid by applying ultrasound (US) treatment to the skin prior to topical application of HA. The contemplated method comprises at least the following process steps: (a) applying ultrasound treatment to a subject's skin surface for about 5 seconds to about 5 minutes; and (b) topically administering a hyaluronic acid solution to the sonicated skin surface; (c) optionally applying an additional ultrasonic treatment to the skin surface for about 5 seconds to about 5 minutes; and (d) optionally, topically administering an additional amount of hyaluronic acid solution to the sonicated skin surface; Thereby facilitating transdermal delivery of hyaluronic acid in a subject.

[0068] In some embodiments, contemplated methods facilitate transdermal delivery of HA with a molecular weight in the range of 300-800 kDa to the epidermis, primarily the upper epidermal layers. Penetration of higher MW HA may also benefit from prior application of US to the treated skin site.

[0069] The inventors have discovered that ultrasound application can be combined with HA complexation (e.g., bringing together HA with a carrier such as a polysaccharide as described herein for GAG transdermal delivery) to increase skin penetration of even higher MW HA and its penetration into even deeper layers of the epidermis.

[0070] An embodiment of the present disclosure is a non-invasive method for enabling or facilitating penetration of high MW hyaluronic acid into deeper layers of the skin in a subject in need thereof, comprising: (a) applying ultrasonic treatment to the skin surface of the subject for about 5 seconds to about 5 minutes; (b) topically administering a complex comprising hyaluronic acid and a polysaccharide to the sonicated skin surface, thereby promoting penetration of hyaluronic acid into the deeper layers of the subject's skin.

[0071] The method is useful for transdermal delivery (ie, delivery through the skin and / or intradermal delivery) of HA with a molecular weight >500 kDa, for example, a molecular weight in the range of 500 kDa to 8000 kDa, 1000 kDa to 5000 kDa, or 500 kDa to 3000 kDa.

[0072] Optionally, steps (a) and (b) of the disclosed method may be repeated at least one or more times (e.g., one, two, three, or more times) as necessary to achieve efficient penetration of hyaluronic acid into deeper layers of the skin; i.e., after the first US application, additional ultrasound treatment may be applied to the treated skin surface for about 5 seconds to about 5 minutes, and then, optionally, an additional amount of hyaluronic acid-polysaccharide complex may be topically administered. In some embodiments, after the second or third US application to the treated skin, additional HA administration is not performed simultaneously.

[0073] In some embodiments, contemplated non-invasive methods for facilitating transdermal delivery of high or low molecular weight hyaluronic acid to deeper layers of the skin are utilized to treat or prevent the skin aging process in a subject in need thereof.

[0074] "Preventing or treating the skin aging process," as used herein, is at least one of maintaining skin hydration, restoring or improving collagen production, slowing the aging process such as wrinkling, or reducing aging indicators related to loss of mechanical properties such as loss of elasticity and skin atrophy.

[0075] Skin atrophy is a common symptom of aging and is often accompanied by ulceration and delayed wound healing. Atrophic skin exhibits decreased HA content and expression of the major cell surface hyaluronan receptor, CD44. With an increasingly aging patient population, managing skin atrophy is becoming a major challenge in the clinic, especially in light of the current lack of effective treatment options.

[0076] In some embodiments, hyaluronic acid is complexed with a quaternized starch (Q-starch) carrier, as defined herein. In some embodiments, Q-starch is a low molecular weight potato starch (26.7 kDa) modified with quaternary amines that forms a self-assembled nano-sized complex with HA (also referred to herein as a "nanocomplex").

[0077] Ultrasound (referred to herein as "US" for simplicity) is a sound wave with a frequency above 18 KHz, which is the limit of human hearing. Ultrasound is a longitudinal wave, i.e., the direction of propagation is the same as the direction of vibration. Ultrasound is also called a "pressure wave" because it causes compression and expansion of the medium, resulting in pressure fluctuations in the medium. The ultrasonic frequency (f) is the number of pressure fluctuation cycles in a medium per unit time (vibration rate) measured in Hertz (Hz), with each cycle consisting of compression and rarefaction. The wave amplitude (A) represents the maximum local pressure measured in Pascals (Pa).

[0078] A typical ultrasonic induction device contains a piezoelectric transducer that converts an electrical signal into ultrasonic waves. By applying an alternating voltage to the piezoelectric material, the material vibrates at the same frequency as the driving current. The transducer can operate in continuous mode (repeated cycles) or pulsed mode (time-separated cycles with signal-free gaps).

[0079] Ultrasound therapy is typically noninvasive and focused. It can be modified by changing various parameters, such as US frequency, intensity, amplitude, acoustic pressure, pulse duration, and period. Therefore, although US waves propagate through multiple tissue layers, they can be focused or targeted to a small volume within a specific organ or tissue for therapeutic purposes. This can lead to tissue heating and destruction in some conditions. The transmitted energy can be concentrated or localized to a specific target or spot within a tissue or organ without adversely affecting the entire tissue or adjacent organs.

[0080] The effects of US on biological tissue primarily include thermal heating, acoustic cavitation, and acoustic streaming. Thermal heating is the result of US waves passing through a medium. US sound waves are absorbed by the medium and induce the formation of heat, which can be conducted, convected, or radiated. The thermal effect increases with frequency and is most pronounced at megahertz frequencies. Low-frequency ultrasound has demonstrated the ability to significantly increase skin permeability, enabling the delivery of various substances through the skin. The primary mechanism explaining ultrasound's ability to increase skin permeability is acoustic cavitation, which can momentarily induce the growth and vibration of air pockets present in the keratinocytes of the stratum corneum.

[0081] The term "cavitation," as used herein, refers to the phenomenon in which rapid changes in pressure in a liquid result in the formation of small vapor-filled cavities where the pressure is relatively low. Expansion cycles in the medium exert negative pressure, pulling molecules apart. When the pressure amplitude exceeds the tensile strength of the liquid in the rarefaction region, small vapor-filled cavities form. When subjected to higher pressures, these cavities, also known as "cavitation bubbles" or "voids," can collapse and generate strong shock waves. Cavitation in liquid media, i.e., the formation of gas cavities, can be the result of US-induced pressure fluctuations in the medium. The cavitation threshold intensity depends on the physical parameters of the medium (temperature, pressure, and dissolved gas concentration) and the acoustic wave.

[0082] The term "acoustic cavitation," as used herein, refers to the formation of bubbles in a medium exposed to ultrasound and the activity (growth, oscillation, or collapse) of existing bubbles. When existing bubbles in a liquid medium are exposed to ultrasound, they oscillate or collapse, generating acoustic radiation; i.e., cavitating bubbles are secondary sources of acoustic sound. There are two types of acoustic cavitation: stable cavitation and inertial cavitation (also known as "transient cavitation"). Stable cavitation is the prolonged oscillation (a significant number of cycles) of bubbles in response to pressure changes. The bubbles expand during the rarefaction phase and contract during the compression phase, oscillating around their equilibrium radius for several cycles. Stable oscillations create a flow of liquid around the bubbles, known as microstreaming, which induces shear stresses. When bubbles are located near biological tissue, such as skin, these shear stresses can cause pore formation and affect tissue permeability.

[0083] Inertial cavitation occurs at higher pressure amplitudes when the pressure amplitude is sufficiently high and reaches a critical value (the inertial cavitation threshold). Bubbles grow violently and collapse; during collapse, symmetric shock waves with high pressures (greater than 10 kbar) and temperatures can be generated in the immediate region of the collapsing bubble. If the bubble is close to a solid surface, collapse can be asymmetric, generating a liquid jet. If collapse occurs near biological tissue, it can cause membrane perforation, reversible pore formation, and / or vascular permeabilization.

[0084] In the embodiments described herein, the acoustic cavitation phenomenon is utilized to increase skin permeability, resulting in transdermal delivery of various GAGs through the skin (for systemic delivery) and between skin layers (for intradermal delivery). Without wishing to be limited by theory, it is hypothesized that cavitation induces disorder in the lipids of the stratum corneum, allowing water to penetrate into the disordered lipid regions and promoting the formation of aquatic channels. These channels in the intercellular lipids of the SC allow the transport of large molecules. Three modes of US-induced cavitation effects are hypothesized: shock waves, microjet impingement on the SC, and microjet penetration into the SC. Both microjets and shock waves may be responsible for the SC permeability-enhancing effect, with microjets being significantly more effective at increasing skin permeability (see, e.g., Tezel and Mitragotri, Biophys J., 2003;85(6):3502-3512; Azagury et al, Adv Drug Deliv Rev., 2014;72:127-143; Wolloch and Kost, J Controlled Release., 2010;148(2):204-211).

[0085] In some embodiments, US is applied in combination with the simultaneous topical application of one or more skin penetration enhancers, such as, but not limited to, surfactants ranging from hydrophobic agents such as oleic acid to hydrophilic sodium lauryl sulfate (SLS). Surfactants are found in many existing therapeutic, cosmetic, and agrochemical preparations and have recently been used to enhance the permeation rate of some drugs via the transdermal route. Surfactants affect the permeability properties of several biological membranes, including the skin. They have the potential to solubilize lipids within the stratum corneum. The penetration of surfactant molecules into the lipid lamellae of the stratum corneum is strongly dependent on the partitioning behavior and solubility of the surfactant.

[0086] The inventors have reported that US (e.g., 3 W / cm 2It was found that simultaneous application of US (0.5 s on and 0.5 s off) and SLS (1% solution) resulted in a change in the pH of the SC, which affected both the structure of the lipid layer and the solubility of SLS inside the skin. Such simultaneous application may result in a synergistic effect of US and SLS on SC permeability.

[0087] In some embodiments, US is applied in combination with SLS to increase skin permeability to GAGs in general and HA in particular.

[0088] Specifically designed and fabricated synthetic microbubbles as cavitation nuclei (cavitation sources) can be used in the contemplated methods. Combining synthetic microbubbles with ultrasound can be utilized to open various biological barriers, including enhancing transdermal delivery of GAGs.

[0089] (Hyaluronic acid and modified starch complex) Aspects of the present disclosure relate to a complex of hyaluronic acid and chemically modified starch.Such complexes, as defined herein, serve as carriers to facilitate the delivery of hyaluronic acid to the deeper layers of the skin.The disclosed complexes are particularly useful for delivering high molecular weight hyaluronic acid.

[0090] Hyaluronic acid has three types of functional groups that can be used for coupling to carrier polymers: anomeric carbonyl, hydroxyl, or carboxyl groups. Depending on the targeted group on hyaluronic acid and the functional groups in the carrier, conjugates can be obtained by direct reaction between both macromolecules. In most cases, modification of hyaluronic acid and / or the carrier polymer is required as a preliminary step to incorporate new functional reactive groups to facilitate conjugation and / or formation of stable complexes. The synthetic strategy for coupling modified polymers to hyaluronic acid is usually selected depending on the functional groups exhibited by the former. Because hyaluronic acid is negatively charged at physiological pH, its complexation with positively charged polymers such as polyaniline, chitosan, poly(β-amino ester), and poly-D-lysine is known and has been used in the synthesis of HA-based nanocarriers. Biodegradable polymers are usually the preferred option.

[0091] The complexes referred to herein include products of conjugation, self-assembly, cross-linking, etc. between HA and carrier polymers, as well as encapsulation of HA by carrier polymers. The complexes contemplated herein are typically nano-sized in size and are also referred to herein as nanocomplexes or nanocarriers.

[0092] The embodiments described herein relate to a complex of hyaluronic acid with quaternized starch (Q-starch), which is obtained by the self-assembly of two polymers. The modified starch is obtained by covalently attaching (i.e., substituting) at least one quaternary amine group to it. Q-starch can be obtained by reacting with 2,3-epoxypropyltrimethylammonium chloride or 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHMAC).

[0093] In some exemplary embodiments, the quaternary amine moiety is (CH3)3-N + -It is.

[0094] Complexes of Q-starch and hyaluronic acid (referred to herein as "Q-starch-HA") are designed and manufactured to feature a desired molar ratio between the positively charged chemical moieties of the modified starch and the negatively charged carboxyl groups of the hyaluronic acid (referred to herein as the "N / O molar ratio," "N / O ratio," or simply "N / O"). The N / O molar ratio influences complex formation and / or transdermal penetration efficacy. Contemplated Q-starch-HAs can be characterized by N / O ratios ranging from about 0.20 to about 3.50, depending, inter alia, on the molecular weight of the hyaluronic acid and / or the type and MW of the Q-starch. For example, N / O may be about 0.20 to about 0.40, about 0.22 to about 0.26, about 0.25 to about 0.35, about 0.30 to about 0.45, about 0.40 to about 0.60, about 0.50 to about 0.70, about 0.65 to about 0.80, about 0.75 to about 0.90, about 0.80 to about 1.00, about 0.85 to about 1.10, or about 1.00 to about 1. 20, about 1.10 to about 1.40, about 1.25 to about 1.50, about 1.35 to about 1.65, about 1.50 to about 1.85, about 1.70 to about 2.00, about 2.10 to about 2.50, about 2.30 to about 2.65, or about 2.60 to about 3.00, and any subranges and individual values ​​therebetween.

[0095] In some embodiments, N / O ranges from about 0.22 to about 0.50, from about 0.25 to about 1.50, or from about 1.00 to about 2.50. In some embodiments, N / O is 0.25.

[0096] (Pharmaceutical Composition) In a further aspect, the present disclosure relates to compositions comprising one or more Q-starch-HA complexes described herein and a physiologically acceptable excipient. The disclosed compositions may be cosmetic compositions, having cosmetic utility and / or pharmaceutical or therapeutic utility. In some embodiments, the compositions are formulated for transdermal administration and include a physiologically acceptable carrier.

[0097] The terms "pharmaceutical composition" and "cosmetic composition," as used herein, refer to compositions essentially comprising at least one Q-starch-HA complex, which may be employed for clinical or cosmetic purposes (such as, but not limited to, therapeutic or anti-aging uses, respectively). A "formulation," as used herein, refers to any mixture of different components or ingredients, at least one of which is a Q-starch-HA complex, prepared in a specific manner, i.e., according to a specific formulation, such that it is suitable for administration to a subject. Such formulations are referred to herein as "Q-starch-HA formulations." For example, Q-starch-HA formulations may be formulated for topical or transdermal administration and may include, for example, one or more Q-starch-HA complexes combined or formulated with one or more carriers, excipients, penetration enhancers, stabilizers, etc.

[0098] As used herein, the terms "pharmaceutically acceptable," "pharmacologically acceptable," and "physiologically acceptable" are used interchangeably and mean approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more specifically, humans. These terms include compounds, molecular entities, excipients, carriers, and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals, or humans, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards, as required, for example, by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA).

[0099] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition or formulation to further facilitate processing and administration of an active ingredient. As used herein, "pharmaceutically acceptable excipient" includes approved preservatives, antioxidants, surfactants (e.g., Tween®-20, Tween®-40, Tween®-60, and Tween®-80), buffers, coatings, isotonicity agents, absorption delaying agents, penetration enhancers, carriers, etc., that are compatible with pharmaceutical administration, do not cause significant irritation to organisms, and do not abolish the biological activity and properties of possible active agents. In liquid formulations, physiologically suitable carriers can be, for example, solvents or dispersion media.

[0100] (kit) In a further aspect, the present invention relates to a kit comprising (a) at least one Q-starch-HA complex or Q-starch-HA formulation as defined herein, (b) a means for applying ultrasound, and (c) optionally, instructions and a means for administering the complexed hyaluronic acid and / or the formulation to a subject in need thereof.

[0101] Contemplated kits are useful for enhancing non-invasive transdermal delivery of hyaluronic acid, e.g., high molecular weight (>1000 kDa) hyaluronic acid, particularly to the deeper layers of the epidermis and / or dermis, which may find use in anti-aging treatments as well as any other treatment modality utilizing hyaluronic acid.

[0102] It is understood that certain features of the present disclosure, 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 present disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or as appropriate, in any other described embodiment of the present disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment would not function without those elements.

[0103] As used herein, the term "about" refers to ±10%.

[0104] The terms "comprises," "comprising," "includes," "including," "having," and conjugates thereof, mean "including but not limited to."

[0105] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0106] Throughout this description, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, a description of a range should be construed as including all specifically disclosed subranges as well as individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be construed as including specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0107] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0108] [Example] Reference is now made to the following examples, which, together with the above description, illustrate some embodiments of the present disclosure in a non-limiting manner. Generally, the nomenclature used herein and the experimental procedures utilized in this disclosure include molecular, chemical, biochemical, and / or microbiological techniques. Such techniques are fully explained in the literature. Other general references are provided throughout this specification. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

[0109] (material) The following materials were purchased from Sigma-Aldrich Inc.: N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS) (P56485); N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC) (E1769); sodium chloride (NaCl) (S-0399); 20(N-morpholino)ethanesulfonic acid hydrate (MES hydrate) (M2933); sodium hydroxide (S-0399); 3-chloro-hydroxypropyltrimethylammonium chloride (348287); phosphate-buffered saline (PBS) (P4417); and sodium lauryl sulfate (SLS) (L5750). Acetone and ethanol were purchased from Bio-Lab. Sodium hyaluronate (1500 KDa; 025693) was purchased from Lifecore Biomedical. Soluble starch (101252) was purchased from Merck. Prolong gold antifade reagent with DAPI (P36935) was purchased from Invitrogen. Full-thickness skin from pig ear was purchased from the Institute of Animal Research, Lahav, Israel. Hylite™ Fluor 647 nm amine (81257) was purchased from Anaspec.

[0110] (i) Hyaluronic acid labeling Labeling of HA was performed as previously described by Sapir et al. (Biomaterials, 2011, 32.7: 1838-1847). The fluorescent dye was covalently attached to HA via carbodiimide chemistry, creating an amide bond between the terminal amine group of the fluorescent molecule and a carboxyl group on the HA. Briefly, 10 ml of a 0.2% (w / v) aqueous HA solution was prepared, and 426 mg of 4-morpholineethanesulfonic acid monohydrate (MES-HO) and 200 mg of NaCl were added to obtain a solution with a pH of 6.5. The mixture was stirred at room temperature for 10 min. The carboxyl groups on HA were activated by the addition of 38.4 mg / 0.5 ml of 1-ethyl(dimethylaminopropyl)-carbodiimide (EDAC) in double-distilled water (DDW), and the coreactant N-hydroxysulfosuccinimide (sulfo-NHS) in DDW at 21.6 mg / 0.5 ml was added to stabilize the reactive intermediate. The mixture was stirred at room temperature for 3 hours, and then 1 mg / 0.5 ml of Hylite™ Fluor 647 amine dye in DDW was added. Stirring was continued for another 12 hours to ensure the formation of an amide bond between the amine group of the dye and the carboxyl group of the HA. The synthesized product (labeled HA) was purified using a dialysis bag with a molecular weight cutoff (MWCO) of 11 kDa and placed in a container containing 5 L of distilled water (DW). The water was replaced with fresh DW six times during the 3-day dialysis period. The dialyzed product was then lyophilized for 72 hours and stored dry at 4°C.

[0111] (ii) Quaternization of starch Modification or derivatization of starch by converting it into a cationic polymer is essential to enable self-assembly with hyaluronic acid through electrostatic interactions between the positively charged groups of the modified starch and the negatively charged carboxyl groups of HA. Quaternary starch (Q-starch), i.e., starch modified by substitution with quaternary ammonium groups, is known as a biocompatible and biodegradable carrier molecule for gene delivery.

[0112] Modification of starch with quaternary amine groups to obtain Q-starch was carried out based on Geresh et al. (Carbohydr Polym. 43(1):75-80, 2000), as previously described by Amar-Lewis et al. (Journal of controlled release 185:109-120, 2014), as outlined in Scheme 1 below. First, 500 mg of water-soluble potato starch (hydrolyzed potato starch, MW 26,765 Da) was dissolved in 10 ml of sodium hydroxide solution (0.19 g / ml) to obtain a starch concentration of 50 mg / ml. The solution was then continuously stirred at room temperature for 30 minutes. Nine grams of the quaternizing reagent, 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHMAC) (9 g, 0.029 mol, 7.8 ml), was dissolved in 20 ml of DW (0.32 g / ml) and added to the starch solution. The reaction mixture was continuously stirred at room temperature for 24 hours. To precipitate the product, 1 volume of product was precipitated by adding 4 volumes of an acidified (1% HCl) mixture of ethanol and acetone (1:3 vol%). The precipitate was washed four times with ethanol, dissolved in a small amount (1-2 ml) of DW, and poured into an 11 kDa cutoff dialysis bag placed in a container containing 5 L of DW. Dialysis was performed to remove unreacted cationic reagent. During the 48 hours of dialysis, the water was replaced with fresh DW four times. The dialyzed product was then lyophilized for 72 hours.

[0113] The quaternization reaction of starch using the quaternizing reagent CHMAC is shown in Scheme 1:

[0114] [ka]

[0115] The quaternization of starch was confirmed by Fourier transform infrared spectroscopy (FT-IR) and elemental analysis (EA). Measurements were taken on a Thermo Nicolet™ FT-IR spectrophotometer (Nicolet™ iS™ 10 FT-IR spectrophotometer). -1and 1478 cm -1 The observation of Q-starch was confirmed by the appearance of strong new absorption bands at 1000 Hz. These bands are associated with the CH and CN stretching vibrations of the quaternary ammonium group (CH3)3N-, respectively. The remaining bands were similar when compared to the FT-IR spectrum of unmodified starch. The samples were prepared in the form of potassium bromide (KBr) pellets.

[0116] The nitrogen content of Q-starch is a necessary parameter because the calculation of the ratio (N / O) of the positively charged amine groups (N) on Q-starch to the negatively charged carboxyl groups (O) on the HA main chain is based on the amount of positive amine groups per starch chain. The weight percentage of nitrogen atoms (N%) of Q-starch was evaluated by the EA method (see, for example, Jeffery et al., Vogel's textbook of quantitative, Chem. Anal., 302-303, 1989) and found to be 3.44%. Calculations based on the quaternization of the 6' position in each glucose monomer of starch suggest that 4.2% is the maximum substitution. The weight percent nitrogen in Q-starch is calculated using Equation 1:

[0117]

number

[0118] The most important advantage of using quaternary amines as substituted molecules is the charge of the polymer, which is largely independent of the pH of the solution. Quaternized starch retains its positive charge over a wide pH range, unlike chitosan conjugates, which are highly pH-dependent and remain stable mainly under acidic conditions.

[0119] (iii) Q starch labeling Q-starch was labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF). First, 100 mg of Q-starch was dissolved in 3 ml of DDW and stirred at room temperature for 30 minutes. The pH was adjusted to 11-12 with 1 M NaOH. After 30 minutes, 7.5 mg of 5-DTAF (dissolved in 0.3 ml of dimethyl sulfoxide (DMSO)) was added to the Q-starch solution and stirred at room temperature for 24 hours in the dark. The reaction mixture was then neutralized with 0.2 M HCl and poured into an 11 KDa cutoff dialysis bag. The labeled polysaccharide, Q-starch-5-DTAF, was separated from free 5-DTAF by extensive dialysis against PBS buffer (pH 7.5) for 72 hours, followed by 48 hours against DDW. The dialyzed product was then lyophilized for 72 hours to obtain purified Q-starch-5-DTAF.

[0120] (iv) Physical properties of HA, Q-starch, and Q-starch-HA complexes The physical properties of Q-starch-HA complexes at different N / O ratios were obtained using zeta potential to measure surface charge, NanoSight to measure diameter size, dynamic light scattering (DLS) to measure hydrodynamic size, and cryo-transmission electron microscopy (cryo-TEM) to measure the size and shape of the complexes.

[0121] (a) Zeta potential (ζ potential) Zeta potential (ζ-potential) is the charge developed at the interface between a solid surface and its liquid medium. This potential, measured in millivolts, can arise through any of several mechanisms. Among these are the dissociation of ionic groups at the particle surface and the differential adsorption of solution ions to the surface region. The net charge at the particle surface affects the ion distribution in the nearby region, increasing the concentration of counterions close to the surface. Thus, an electric double layer forms in the region of the particle-liquid interface, consisting of two parts: an inner region containing ions relatively tightly bound to the surface, and an outer region where the balance between electrostatic forces and random thermal motion determines the ion distribution. Therefore, the potential in this region decays with increasing distance from the surface, conventionally considered to be zero, until it reaches the bulk solution value at a sufficient distance.

[0122] In an electric field, each particle and its most closely associated ions move through the solution as a unit, and the potential at the surface of the shear between this unit and the surrounding medium is known as the zeta potential. In other words, the zeta potential is defined as the average electrostatic potential that exists at the hydrodynamic surface of the shear. When a layer of macromolecules is adsorbed onto the surface of the particle, it shifts the shear plane further away from the surface, changing the zeta potential.

[0123] Measurement of the ζ-potential is currently the simplest and most straightforward method for characterizing the surface of charged colloids and is most relevant for the practical study and control of colloid stability and aggregation processes.

[0124] The surface charge of hyaluronic acid, Q-starch, and Q-starch-HA complexes was measured by zeta potential measurement using a Zetasizer (ZN-NanoSizer, Malvern, England). Complexes were prepared at different N / O ratios (e.g., 0.25–3) and diluted to a final HA concentration of 26 mM in a volume of 1 ml of DDW, as described in Example 1 below. Samples were transferred to U-tube cuvettes (DTS1070, Malvern) and measured in automatic mode at 25°C. The Smoluchowski model (Smoluchowski, Z. Phys. Chem., 1917, 92:129–168) was used to calculate the zeta potential. For each sample, the zeta potential value was expressed as the average of three runs (triplicates ± standard deviation).

[0125] (b) Dynamic light scattering (DLS) Dynamic light scattering (DLS) measures the temporal fluctuations of light scattered by particles due to their Brownian motion when a solution containing particles is placed in the path of a monochromatic beam of light. The Brownian motion of a particle correlates with its hydrodynamic diameter. The smaller the particle, the faster it can diffuse. DLS is also known as photon correlation spectroscopy or quasi-elastic light scattering. This technique analyzes the modulation of the intensity of scattered light as a function of time, providing information about particle size in terms of hydrodynamic diameter. DLS is a sensitive, non-invasive, and powerful analytical tool routinely used for the characterization of macromolecules, colloids, and nanoparticles in solution.

[0126] The hydrodynamic size (radius) distribution of the complexes disclosed herein was measured by DLS. Complexes were prepared at different N / O ratios (e.g., 0.25 to 3) and diluted to a final HA concentration of 100 mM in DDW in a volume of 260 μl, as described in Example 1 below. Spectra were collected using a CGS-3 (ALV, Langen, Germany) goniometer at 20 mW laser power on the He-Ne laser line (632.8 nm). Autocorrelation functions (correlograms) were calculated with an ALV / LSE5003 correlator at a 90-degree angle and a temperature of 25°C for 30-second time windows (10 in total). Autocorrelation functions were fitted using the CONTIN program (Provencher, Since Direct, 1982, 27: 229-242).

[0127] (c) Nanosite NanoSight analysis is a technique capable of sizing and quantifying nanoparticles through the use of light scattering. Unlike conventional DLS, it uses a charge-coupled device (CCD) camera to track the movement of individual nanoparticles in real time, and the system derives their hydrodynamic radius through the Stokes-Einstein equation. NanoSight also goes beyond DLS in that it explicitly quantifies particles smaller than 1 micron and can more accurately characterize polydisperse samples. When illuminated by laser light, the NanoSight nanoparticle analysis instrument generates videos of ensembles of nanoparticles moving under Brownian motion in a liquid. Within a specially designed and constructed laser illumination device mounted below the microscope objective, particles in a liquid sample passing through the beam path are seen by the instrument as tiny dots of light rapidly moving under Brownian motion. This capability of the NanoSight system allows for dynamic analysis of the paths particles take under Brownian motion over a suitable time period (e.g., 30 seconds).

[0128] The diameter of Q-starch / HA complexes in aqueous solution was determined using NanoSight technology. Complexes were prepared at N / O molar ratios of 0.25 to 3 and diluted to a final concentration of 13 mM in a final volume of 2 ml of DDW, as described in Example 1 herein. A NanoSight NS300 instrument (Malvern Instruments, Malvern, UK) equipped with a 642 nm laser module and a 650 nm long-pass filter was used. All measurements were performed at room temperature in a flow cell (software: NTA3.1(iss2)). All samples were analyzed under a 20x objective, and 60-second video clips were recorded.

[0129] (d) Cryo-transmission electron microscope (Cryo-TEM) Cryogenic transmission electron microscopy (Cryo-TEM), also known as Cryo-EM, is a form of cryogenic electron microscopy, more specifically, a type of transmission electron microscope (TEM) in which samples are studied at cryogenic temperatures. The cryogenic temperature range is defined as -150 °C (-238 °F) to absolute zero (-273 °C or -460 °F), approaching the temperature at which molecular motion theoretically ceases as completely as possible, and materials at low temperatures approach as close to a static, highly ordered state as possible. These extreme conditions alter material properties such as strength, thermal conductivity, ductility, and electrical resistance.

[0130] In a transmission electron microscope, accelerated electrons pass through and interact with a specimen. Interference between scattered and unscattered electrons results in so-called phase contrast and image formation. Because electron microscopes require a high vacuum, live cells or, more generally, hydrated specimens cannot be examined by this method at room temperature. In cryo-TEM, this problem is solved by embedding the specimen in amorphous ice through plunge freezing in liquid ethane. When imaged at cryogenic temperatures (e.g., -178 °C), the vapor pressure of so-called vitrified specimens is low, and therefore, specimens can be imaged in their hydrated state. The utility of transmission electron cryo-microscopy derives from the fact that it allows the observation of specimens that have not been stained or fixed in any way, showing them in their native environment. Cryo-TEM provides the determination of macromolecular structures at near-atomic resolution.

[0131] The size and shape of HA, Q-starch, and Q-starch-HA complexes in solution were visualized and characterized by direct imaging of aqueous solutions using cryo-TEM. Complexes were prepared at a final HA concentration of 260 mM in 40 μl of DDW with 0.25 N / O as described in Example 1. A 2.5 μl drop of the solution was placed on a carbon lacey film supported on a 300-mesh Cu grid (PELCO® TEM, Ted Pella Ltd). Excess liquid was blotted off, and the samples were vitrified by rapidly plunging into liquid ethane pre-cooled with liquid nitrogen in a controlled-environment automated vitrification system (Leica EM GP) with controlled temperature and relative humidity. Samples were cryo-TEMped in an FEI Tecnai™ G cryo-TEM operated at 120 kV and equipped with a Gatan 626 cold stage control unit. 2 The samples were examined using a 12TWIN transmission electron microscope at −178°C. 2D images were captured with a Gatan 794 MultiScan charge-coupled device (CCD) camera.

[0132] (v) Skin treatment For in vitro experiments, full-thickness skin from pig ears (from behind the ear) was used. The skin was separated from the ear using a scalpel, cut into 2 × 2 cm pieces, and stored frozen (-20 °C) until use. Before each experiment, the skin samples were thawed to room temperature for 10 min.

[0133] (vi) Skin conductivity measurement Skin integrity and the effects of ultrasound pretreatment were assessed by skin conductivity measurements. Ag / AgCl 4 mm disk electrodes were inserted into both diffusion cell compartments in in vitro experiments. An AC voltage of 200 mV at 10 Hz was applied in vitro using a function generator (Agilent 33120A, Palo Alto, CA, USA). Current was measured using a multimeter (Fluke 45 display multimeter, Everett, WA, USA).

[0134] (vii) In vitro skin permeability measurement method and device Skin permeability measurements were performed in a vertical static glass diffusion cell consisting of a donor compartment and a receiver compartment. The skin was placed between two separate compartments, with the stratum corneum (SC) facing the donor compartment. The donor compartment was filled with 6 ml of 1% sodium lauryl sulfate (SLS; a surfactant commonly used to enhance the efficacy of topically applied formulations) in PBS. For ultrasound-treated skin samples, ultrasound (QSonica Q700 Sonicator, frequency = 20 kHz, 8.2 W / cm) was applied. 2 (3% amplitude, probe diameter 1.3 cm) was applied as a pretreatment: the ultrasound probe was placed in the donor compartment, 8 mm above the skin surface. To minimize thermal effects, a 50% duty cycle mode was selected (i.e., 1 second on, 1 second off), and the contents of the donor compartment were replaced with fresh room temperature medium every 30 seconds (these ultrasound parameters were used because they were found to be optimal in previous studies). To assess skin permeability, conductivity measurements were performed at the beginning of the experiment, before and during US exposure. A conductivity of 0.7 (kΩ*cm) was used. 2 ) -1Skin with higher conductivity was considered defective and was not used. The US was turned off after 5 min of exposure, during which time all skin samples reached conductivities 50–60 times higher than their initial conductivity.

[0135] After US pretreatment, the skin was removed from the diffusion cell, washed with PBS, and placed back on the cell. 700 μl of fluorescently labeled HA (HA) at the desired N / O molar ratio (e.g., 0.25, HA concentration 260 mM) was added. Hylite Fluor6 ) or 700 μl of Q-starch-HA complex (HA is labeled (Q-starch-HA Hylite Fluor6 ) or both Q-starch and HA are labeled (Q-starch 5-DTAF -HA Hylite Fluor647 ) was placed on the skin for 24 hours, after which the skin samples were fixed in 4% paraformaldehyde and embedded in paraffin. After deparaffinization, 5 μm-thick sections from each sample were cut, placed on slides, and stained for histological analysis. The slides were rehydrated and visualized using a confocal laser scanning microscope. Confocal fluorescence images were acquired using a ZEISS (Germany) Airyscan confocal system on an LSM-880 with a plan-apochromat 20× / 0.8 DIC M27 objective.

[0136] To visualize skin autofluorescence, excitation was performed with a 488 nm argon laser and emission was detected at a range of 490 to 597 nm. To visualize HA labeled with Hylite™ Fluor 647, excitation was performed with a 633 nm HeNe laser and emission was detected at a range of 638 to 759 nm.

[0137] (viii) Histological analysis (DAPI staining) DAPI (4',6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain that exhibits approximately 20-fold fluorescence enhancement upon binding to AT regions of dsDNA. It is excited by a violet (405 nm) laser. DAPI can pass through intact cell membranes and can be used to stain both live and fixed cells. For DAPI staining, skin tissues were fixed in 4% formalin and embedded in paraffin wax. After deparaffinization, the tissues were sectioned at 5 μm thickness using a microtome (Leica RM2255 microtome, England) and rehydrated. Before staining, slides were first washed with xylene (twice for 10 min each), then 100% ethanol (twice for 10 min each), 95% ethanol (5 min), 70% ethanol (5 min), 50% ethanol (5 min), distilled water (5 min), and PBS (twice for 10 min). After washing, slides were mounted with DAPI for nuclear visualization.

[0138] Example 1 Formation and Characterization of Q-Starch-HA Complexes Q-starch-HA conjugates were prepared with different molar ratios (N / O molar ratios) between the positively charged amine groups (N) of Q-starch and the negatively charged carboxyl groups (O) of the main chain of HA. The amount of HA (X mg of HA) was predetermined for each measurement, and the amount of O (mol O) of carboxyl groups on HA was calculated according to Equation 2:

[0139]

number

[0140] The amount of Q-starch required for the desired N / O was calculated using Equation 3:

[0141]

number

[0142] To prepare the Q-starch-HA complex, stock solutions of Q-starch (or labeled Q-starch) and HA (or labeled HA) were first prepared. For example, 3 mg of Q-starch was dissolved in 7.5 ml of DDW at a concentration of 0.4 mg / ml, and 1 mg of HA was dissolved in 1 ml of DDW at a concentration of 0.1 mg / ml. The amount of Q-starch (or Q-starch-5-DTAF) for a specific desired N / O ratio was calculated based on the nitrogen content (weight % N) using Equation 3. The desired amounts of Q-starch and HA solutions were collected to prepare the complex. After gentle vortexing, the sample was incubated at room temperature for 40 minutes to allow complex formation by self-assembly. The Q-starch-HA complex was prepared in an Eppendorf flask, so that the amount of HA was kept constant while the amount of added carrier (Q-starch) was varied to obtain the desired N / O ratio. The HA concentration in each Eppendorf flask containing a final volume of 700 μl of complex solution was 260 mM HA.

[0143] The size, surface charge, and morphology of Q-starch / HA complexes are very important parameters because their values ​​can affect their penetration into the skin. The physical properties of Q-starch-HA complexes at different N / O ratios were obtained using zeta potential to measure surface charge, nanoscopy for diameter size, dynamic light scattering (DLS) for hydrodynamic size measurement, and cryo-transmission electron microscopy (cryo-TEM) for the size and shape measurement of the complexes, as described in Materials and Methods.

[0144] (i) Characterization of the complex by dynamic light scattering (DLS) and nanosight Dynamic light scattering (DLS) and nanosight techniques were used to determine the average hydrodynamic radius and diameter of Q-starch / HA complexes. Using dynamic light scattering, scattered light interacting with the complexes suspended in a medium was measured, and its diffusion coefficient was calculated. Their hydrodynamic radii were then calculated using the Stokes-Einstein equation. The size distributions of Q-starch-HA complexes at increasing N / O ratios are shown in Figure 1A. As can be seen, the average hydrodynamic radius for all evaluated N / O molar ratios (0.25–3) was approximately 100 nm, with no significant differences. Furthermore, the size distributions of complexes for each N / O ratio were relatively uniform in size, with peaks all within the same range. However, DLS measurements of free HA and free Q-starch demonstrated a very wide range of hydrodynamic radius sizes, as shown in Figure 1B. These size distributions clearly indicate a lack of evidence of internal self-interactions to form particles, in contrast to the complexes, which exhibited narrow size distributions.

[0145] To verify the DLS results, the size diameter of the Q-starch / HA complex was evaluated by another method, NanoSite. Figure 2 shows representative results for free HA, free Q-starch, and the Q-starch / HA complex with an N / O ratio of 0.25. It can be seen that both the free HA plot and the free Q-starch plot exhibited a very broad size distribution with many unclear peaks, whereas the complex plot exhibited a relatively narrow size distribution. When measured by NanoSite, no significant differences were observed in the size distribution (average diameter) of the Q-starch / HA complex with increasing N / O ratios (results not shown), which is consistent with the results obtained from DLS measurements.

[0146] (ii) Characterization of the zeta potential complex Zeta potential is a function of the particle's surface charge, any adsorbed layers at the interface, and the nature and composition of the surrounding suspension medium. To determine the surface charge of the complexes at different N / O molar ratios, zeta potential measurements were performed on Q-starch-HA complexes with different N / O ratios, as well as on free HA and freshly prepared uncomplexed Q-starch. The results are shown in Figure 3. As expected, the negatively charged carboxyl groups of this polymer resulted in a negative zeta potential value for uncomplexed HA (-70 mV), while a very positive zeta potential value of 42 mV was obtained for uncomplexed Q-starch, confirming the presence of positively charged quaternary amine groups. As shown in Figure 3, increasing the N / O ratio resulted in an increase in the zeta potential value from a negative value of approximately -36 mV for N / O 0.25 to a positive value of approximately 40 mV for N / O 3.

[0147] (iii) Characterization of the complex by cryo-transmission electron microscopy (Cryo-TEM) The macromolecular structure or geometry of free HA, free Q-starch, and the Q-starch-HA complex (HA and Q-starch were the same amount as the Q-starch-HA complex) in different N / O conditions was determined using cryo-TEM techniques as described in Materials and Methods. The results are shown in Figure 4A-G. As shown in Figure 4A, the free Q-starch sample could not be clearly visualized by cryo-TEM; only a clean grid was observed. This can be explained by the fact that the polymer in aqueous solution is sufficiently dissolved and has low electron density, so the diffusing atoms cannot be seen separately. The same results were observed for free HA (Figure 4B). In contrast, the cryo-TEM images of the Q-starch / HA complex (Figure 4C-G) showed mostly small, spherical, condensed aggregates.

[0148] The stability of Q-starch-HA complexes in aqueous solution over time was assessed by evaluating their hydrodynamic size 3, 24, and 48 hours after their formation. For example, complexes characterized by N / O 0.25 were evaluated by nanoscopy and cryo-TEM, and although their size increased slightly over time (presumably due to HA swelling in aqueous media), their diameter did not double or increase significantly, clearly indicating that aggregates of the complexes did not form, i.e., the complexes were stable (results not shown).

[0149] Example 2 (Effect of ultrasound irradiation on skin permeability of hyaluronic acid solution) The in vitro uptake of a solution of fluorescently labeled high molecular weight (HMW) HA by porcine skin samples was measured with or without prior application of low-frequency ultrasound to the skin samples using the diffusion cell method and apparatus described in Materials and Methods. Prior to each permeability measurement, a 0.3% (w / v) HA solution was prepared by dissolving 3.6 mg of HA (Mw 1500 KDa) in 1.2 ml of DDW in a glass vial and stirring at room temperature until completely dissolved. HA was fluorescently labeled with Hylite™ Fluor 647 amine dye (referred to herein as HA) as described in Materials and Methods. Hylite Fluor647 Before applying the labeled HA to the skin samples, they were subjected to 5 minutes of US application (20 KHz, 8.2 W / cm) as described in Materials and Methods. 2 A pretreatment with US (duty cycle = 50%) was performed. Confocal microscopy images of an exemplary pig ear skin section 24 hours after US application are shown in Figures 5A-5C. The fluorescence intensity of labeled HA as a function of distance from the SC to a depth of 200 μm, calculated for the pixels of an exemplary rectangular cross-section in image j, is shown in each graph.

[0150] As shown in Figure 5A, without US pretreatment, all fluorescence was confined to the SC layer, clearly indicating that HA did not penetrate the skin. On the other hand, in the skin samples pretreated with US, the fluorescence intensity was higher in the epidermis and scattered down to a depth of 50 μm into the SC layer (Figure 5B). These results confirm that US application can affect skin penetration beyond the SC layer.

[0151] One possible explanation for this phenomenon of enhanced SC permeability is mechanical effects caused by US application, such as cavitation. As discussed herein, both microjets and shock waves may be involved in the enhancement of SC permeability.

[0152] However, HA barely penetrated into the dermis, the target layer for HA biological activity, which can be explained by the fact that HMW HA is a large molecule that slows down its diffusion through the skin, especially in aqueous solutions.

[0153] Example 3 (Effect of complex formation between hyaluronic acid and quaternary starch on its skin permeability) To facilitate HA penetration into the deep dermis, the hydrodynamic size of HA was condensed and its radius reduced to a permeable size by complexing negatively charged HA with a cationic carrier through self-assembly. Complexing HA with a carrier has the added benefit of extending the HA half-life, thus providing HA with longer stability and retention time in the skin. In this study, a positively charged modified starch (Q-starch) was used as the HA carrier.

[0154] The penetration of Q-starch-HA complexes into deeper skin layers after topical application to pig ear skin samples was studied in vitro using the diffusion cell method and apparatus described in Materials and Methods. HA was treated with Hylite™ Fluor 647 amine dye (HA). Hylite Fluor647 ) and the complex, Q-starch-HA Hylite Fluor647The complex was formulated at an N / O molar ratio of 0.25 and topically applied to skin samples for 24 hours, with or without ultrasound preapplication (5 minutes). Untreated ear skin sections (without US application and complex administration) served as a control group. This control group was used to evaluate autofluorescence at various depths or layers of the skin samples. Visualization of histologically stained treated and untreated (control) pig skin cross sections was performed by confocal microscopy (excitation was performed with a 633 nm HeNe laser, and emission was detected between 638 nm and 759 nm). Exemplary confocal and bright-field images of treated skin are shown in Figures 6A-6D. In DAPI-stained cross sections, the complexed HA Hylite Fluor647 The complexed HA appeared as red staining and the cell nuclei were stained blue. Hylite Fluor647 appeared pinkish red. Q-starch-HA Hylite Fluor647 Fluorescence intensity of the complex Q-starch-HA as a function of distance from the SC to a depth of 350 μm, calculated (by image j), per pixel of any skin cross section indicated by a rectangle in the image. Hylite Fluor647 The fluorescence intensity of the complex is presented for both US-pretreated and non-pretreated skin. For convenience, the fluorescence intensity calculated for any pixel is referred to herein as the "pixel fluorescence intensity."

[0155] As can be seen in Figures 6A and 6B, in skin that was not pretreated with US before complexed HA application, the topically administered complex remained mostly in the SC layer, the top layer of skin. As calculated, pixel fluorescence intensity was higher in the SC layer than in the epidermis and dermis (although some fluorescence was detected in these deeper layers). In contrast, Q-starch-HA Hylite Fluor647 In skin samples pretreated with US for 5 min before topical application of the complex 24 h later, the complex penetrated the SC barrier into the epidermis, including the basal cell layer of the dermis (Figures 6C-6D). As seen in Figure 6D, pixel fluorescence intensity was higher in the epidermal and dermal layers than in the SC layer.

[0156] To quantify the difference in fluorescence intensity of Q-starch / HA complex in the deeper skin layers between the US-pretreated skin group versus the non-US-pretreated skin group, three groups of skin samples were observed: (i) labeled Q-starch-HA complex (Q-starch-HA Hylite Fluor647 (ii) skin samples treated with ultrasound for 5 minutes and then treated with Q-starch-HA Hylite Fluor647 (iii) a control group—skin samples treated with neither ultrasound nor the labeled complex for 24 hours; and (iv) a control group—skin samples treated with neither ultrasound nor the labeled complex. For each skin sample, three randomly selected rectangular cross sections of the layers were used to calculate the pixel fluorescence intensity. The results are shown in Figure 7.

[0157] As shown in Figure 7, skin pretreated with US exhibited higher fluorescence intensity in the epidermis and dermis compared to skin not pretreated by US application.

[0158] As further shown in Figure 7, in the SC layer (0–20 μm), the autofluorescence measured in the control group was significantly higher than that measured in both US-pretreated and non-pretreated skin. Hylite Fluor647 However, in the epidermis (20-100 μm), the difference in fluorescence intensity between skin not pretreated with US and autofluorescence was dramatically smaller, and in the dermis (100-2000 μm), autofluorescence was significantly lower than that of HA. Hylite Fluor647 On the other hand, in the skin pretreated with US, in contrast to the skin not pretreated with US, the fluorescence of HA Hylite Fluor647 The fluorescence intensity of was significantly higher than the autofluorescence of both the epidermis and dermis, clearly indicating that the complex had indeed penetrated these layers.

[0159] From these results, it can be concluded that the combination of ultrasound preapplication and a carrier for delivering HA resulted in highly effective penetration of HA into the deeper layers of the skin, including the target layer (dermis).

[0160] Example 4 (Stability of Q-starch / HA complex in the skin layer) To assess the stability of the Q-starch / HA complex in the skin layers under the treatment conditions described in Example 3 above, the carrier Q-starch was labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (green), the HA was labeled with Hylite™ Fluor 647 (red), and the complex Q-starch with a final concentration of 0.25 N / O and 260 mM complexed HA was prepared. 5-DTAF -HA Hylite Fluor647 s were prepared as described in Materials and Methods. Fixed samples were stained with DAPI to assess intact nucleated cells in the skin layer below the SC (SC contains dead, anucleated cells). Stained samples were sectioned and visualized using a confocal microscope.

[0161] Confocal images of pig skin samples were acquired 24 hours after topical administration of the labeled complex, either with or without a preceding 5-minute US application. Exemplary confocal images are shown in Figures 8A-8D.

[0162] As can be seen in Figures 8A-8B, without US pretreatment, Q-starch at 24 h 5-DTAF -HA Hylite Fluor647 Topical administration of Q-starch resulted in both green and red staining present in the SC in a similar pattern. 5-DTAF Green staining due to the presence of HA Hylite Fluor647 The green and red staining patterns are similar (Figures 8C-8D). These results indicate that HA does not disassemble from Q-starch and that the complex retains its stability in the deep skin layers.

[0163] Example 5 (In vivo permeability test) The following study is designed to evaluate the dermal uptake of Q-starch-hyaluronic acid complex in mice in vivo.

[0164] Mice were divided into the following groups: Group I: control - mice that did not receive any treatment; Group II: mice treated with Q-starch-HA without US pretreatment; and Group III: mice that received US pretreatment followed by administration of Q-starch-HA complexes (at different N / O molar ratios). Hyaluronic acid was grafted onto the HA matrix using Hylite™ Fluor 647 amine dye (HA). Hylite Fluor647 ) and contacted with Q-starch to form Q-starch-HA Hylite Fluor647 The following steps in the research protocol are applied: 1. Shave the back of each mouse using hair clippers, then anesthetize the mouse with isoflurane.

[0165] 2. Attach the rubber ring with the plastic cylinder to the top of the shaved skin using biological adhesive and pour 2 ml of PBS into the inside of the chamber.

[0166] 3. An approximately 1 cm incision is made near the tail. One conductive electrode is placed inside the chamber and another inside the incision to measure the initial skin conductivity.

[0167] 4. The PBS in the chamber is then replaced with 1% SLS in PBS and the skin conductivity is measured again.

[0168] 5. Mice in group III were subjected to US treatment (QSonica Q700 Sonicator, frequency = 20 kHz, 6.1-10.5 W / cm 2 A pretreatment was performed: the ultrasound probe was placed in a plastic cylinder 8 mm from the skin surface. To minimize thermal effects, a 50% duty cycle mode was selected (i.e., 0.5 seconds on, 0.5 seconds off), and the contents of the plastic cylinder were replaced with fresh medium every 20-30 seconds (depending on the temperature). To assess skin permeability, conductivity measurements were performed during ultrasound exposure. Ultrasound application was stopped when the conductivity reached 50-70 times the initial conductivity, or 0.70 (kΩ*cm). 2 ) -1When the predetermined value is reached, it turns off.

[0169] 6. For mice ingroups II and III, the plastic cylinder was removed and Q-starch-HA was placed on the mice. Hylite Fluor647 The complex is prepared 40 minutes before US application and placed inside a rubber ring. A parafilm cover is applied to prevent fluid leakage from the ring.

[0170] 7. 20 hours after administration of the complex, the mice of groups I-III are sacrificed, the skin is removed, fixed in 4% formalin and cut into 5 μm slices (slides).

[0171] 8. Rehydrate the slides and visualize using a confocal laser scanning microscope.

[0172] Example 6 (In vivo model of UV radiation-induced wrinkles) To create a skin aging model in mice, it is necessary to induce collagen degradation in the dermal layer. Therefore, as a first step, this degradation is induced by ultraviolet (UV) radiation, which is carried out over a period of approximately 5-12 weeks, until wrinkles appear and collagen fibers decrease.

[0173] Epidermal and dermal thickness are assessed by light microscopy, and skin elasticity and skin hydration are measured by different instruments, with the reasonable expectation that elasticity and skin hydration will decrease. Hylite Fluor647 The effect of complex application on the appearance of mouse skin, with or without US pretreatment, was analyzed and examined by histological staining. For histological analysis, skin samples must maintain their structure and function as they would in an animal body. Therefore, samples undergo several steps: fixation, embedding, sectioning, and staining. Hematoxylin and eosin (H&E) staining is performed to assess the thickness of the epidermis and dermis. [Brief explanation of the drawings]

[0174] [Figure 1A]Figures 1A-1B show the size distributions of a complex of quaternary starch (Q-starch) with hyaluronic acid (HA) (Q-starch-HA complex) (1A) and free Q-starch and HA (1B) obtained using dynamic light scattering (DLS). The Q-starch-HA complex is characterized by an increased ratio between the positively charged amine groups (N) of Q-starch and the negatively charged carboxyl groups (O) of the HA backbone (N / O ratio). [Figure 1B] Figures 1A-1B show the size distributions of a complex of quaternary starch (Q-starch) with hyaluronic acid (HA) (Q-starch-HA complex) (1A) and free Q-starch and HA (1B) obtained using dynamic light scattering (DLS). The Q-starch-HA complex is characterized by an increased ratio between the positively charged amine groups (N) of Q-starch and the negatively charged carboxyl groups (O) of the HA backbone (N / O ratio). [Figure 2] FIG. 2 is a graph showing the size distribution (mean diameter) of free Q-starch, free HA, and Q-starch-HA complexes with an N / O of 0.25, as measured using the NanoSight system. [Figure 3] FIG. 3 is a bar graph showing the average zeta potential (a function of particle surface charge) of free HA, Q-starch, and Q-starch-HA complexes characterized by increasing N / O ratios. [Figure 4A] Figures 4A-4G show exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and freshly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C-4G). [Figure 4B] Figures 4A-4G show exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and freshly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C-4G). [Figure 4C]Figures 4A-4G show exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and freshly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C-4G). [Figure 4D] Figures 4A-4G show exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and freshly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C-4G). [Figure 4E] Figures 4A-4G show exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and freshly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C-4G). [Figure 4F] Figures 4A-4G show exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and freshly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C-4G). [Figure 4G] Figures 4A-4G show exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and freshly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C-4G). [Figure 5A]Figures 5A-5B are bright-field confocal images of exemplary pig ear skin cross sections after 24 hours of incubation with 0.3% (w / v) HA labeled with Hylite™ Fluor 647 dye (HAHylite Fluor 647) without ultrasound (US) pretreatment (5A) or after 5 minutes of US application (5B). The stratum corneum (SC), epidermal layer, and dermal layer are shown (bar: 20 μm). The fluorescence intensity of HAHylite Fluor 647 calculated for pixels in the exemplary rectangular cross section shown by image j as a function of distance from the SC to a depth of 200 μm is shown for each cross section. The vertical dashed lines represent the separation between layers. The horizontal dashed lines indicate skin autofluorescence at the wavelength of the labeled HA. [Figure 5B] Figures 5A-5B are bright-field confocal images of exemplary pig ear skin cross sections after 24 hours of incubation with 0.3% (w / v) HA labeled with Hylite™ Fluor 647 dye (HAHylite Fluor 647) without ultrasound (US) pretreatment (5A) or after 5 minutes of US application (5B). The stratum corneum (SC), epidermal layer, and dermal layer are shown (bar: 20 μm). The fluorescence intensity of HAHylite Fluor 647 calculated for pixels in the exemplary rectangular cross section shown by image j as a function of distance from the SC to a depth of 200 μm is shown for each cross section. The vertical dashed lines represent the separation between layers. The horizontal dashed lines indicate skin autofluorescence at the wavelength of the labeled HA. [Figure 6A]Figures 6A-6D show confocal images of representative pig ear skin cross sections stained histologically after 24 hours of incubation with a labeled Q-starch / HA complex (Q-starch-HA Hylite Fluor 647) characterized by an N / O molar ratio of 0.25. Skin samples were either not pretreated with ultrasound prior to topical application of the labeled Q-starch / HA complex (6A, 6B) or were treated with US for 5 minutes prior to complex application (6C, 6D). Figure 6A and Figure 6C are confocal images showing intact nucleated cells in the skin layers below the stratum corneum (SC) (nuclear staining with 4',6-diamidino-2-phenylindole (DAPI)); Figure 6B and Figure 6D are bright-field confocal images. The SC, epidermal, and dermal layers are shown (bar: 20 µm). The fluorescence intensity of Q-Starch-HAHylite Fluor 647 was calculated for pixels in the exemplary rectangular cross-section shown by image j as a distance from the SC to a depth of 350 µm. The fluorescence intensity of Q-Starch-HAHylite Fluor 647 is shown for each cross-section. The vertical dashed lines represent the separation between the layers. The horizontal dashed lines indicate the autofluorescence of the skin at the wavelength of the labeled HA. [Figure 6B]Figures 6A-6D show confocal images of representative pig ear skin cross sections stained histologically after 24 hours of incubation with a labeled Q-starch / HA complex (Q-starch-HA Hylite Fluor 647) characterized by an N / O molar ratio of 0.25. Skin samples were either not pretreated with ultrasound prior to topical application of the labeled Q-starch / HA complex (6A, 6B) or were treated with US for 5 minutes prior to complex application (6C, 6D). Figure 6A and Figure 6C are confocal images showing intact nucleated cells in the skin layers below the stratum corneum (SC) (nuclear staining with 4',6-diamidino-2-phenylindole (DAPI)); Figure 6B and Figure 6D are bright-field confocal images. The SC, epidermal, and dermal layers are shown (bar: 20 µm). The fluorescence intensity of Q-Starch-HAHylite Fluor 647 was calculated for pixels in the exemplary rectangular cross-section shown by image j as a distance from the SC to a depth of 350 µm. The fluorescence intensity of Q-Starch-HAHylite Fluor 647 is shown for each cross-section. The vertical dashed lines represent the separation between the layers. The horizontal dashed lines indicate the autofluorescence of the skin at the wavelength of the labeled HA. [Figure 6C]Figures 6A-6D show confocal images of representative pig ear skin cross sections stained histologically after 24 hours of incubation with a labeled Q-starch / HA complex (Q-starch-HA Hylite Fluor 647) characterized by an N / O molar ratio of 0.25. Skin samples were either not pretreated with ultrasound prior to topical application of the labeled Q-starch / HA complex (6A, 6B) or were treated with US for 5 minutes prior to complex application (6C, 6D). Figure 6A and Figure 6C are confocal images showing intact nucleated cells in the skin layers below the stratum corneum (SC) (nuclear staining with 4',6-diamidino-2-phenylindole (DAPI)); Figure 6B and Figure 6D are bright-field confocal images. The SC, epidermal, and dermal layers are shown (bar: 20 µm). The fluorescence intensity of Q-Starch-HAHylite Fluor 647 was calculated for pixels in the exemplary rectangular cross-section shown by image j as a distance from the SC to a depth of 350 µm. The fluorescence intensity of Q-Starch-HAHylite Fluor 647 is shown for each cross-section. The vertical dashed lines represent the separation between the layers. The horizontal dashed lines indicate the autofluorescence of the skin at the wavelength of the labeled HA. [Figure 6D]Figures 6A-6D show confocal images of representative pig ear skin cross sections stained histologically after 24 hours of incubation with a labeled Q-starch / HA complex (Q-starch-HA Hylite Fluor 647) characterized by an N / O molar ratio of 0.25. Skin samples were either not pretreated with ultrasound prior to topical application of the labeled Q-starch / HA complex (6A, 6B) or were treated with US for 5 minutes prior to complex application (6C, 6D). Figure 6A and Figure 6C are confocal images showing intact nucleated cells in the skin layers below the stratum corneum (SC) (nuclear staining with 4',6-diamidino-2-phenylindole (DAPI)); Figure 6B and Figure 6D are bright-field confocal images. The SC, epidermal, and dermal layers are shown (bar: 20 µm). The fluorescence intensity of Q-Starch-HAHylite Fluor 647 was calculated for pixels in the exemplary rectangular cross-section shown by image j as a distance from the SC to a depth of 350 µm. The fluorescence intensity of Q-Starch-HAHylite Fluor 647 is shown for each cross-section. The vertical dashed lines represent the separation between the layers. The horizontal dashed lines indicate the autofluorescence of the skin at the wavelength of the labeled HA. [Figure 7] Figure 7 is a bar graph showing the fluorescence intensity measured at different wavelengths for labeled HA (HAHylite Fluor 647) in three layers of pig ear skin: SC (0–20 μm), epidermis (20–100 μm), and dermis (100–2000 μm). Three groups of skin samples were observed: (i) skin samples to which labeled Q-starch-HA complex (Q-starch-HAHylite Fluor 647) was topically applied for 24 h; (ii) skin samples treated with ultrasound for 5 min and then topically applied Q-starch-HAHylite Fluor 647 for 24 h; and (iii) a control group—skin samples not treated with either ultrasound or the labeled complex. This group served for autofluorescence measurements. Fluorescence intensity was calculated by ImageJ based on data recorded from confocal scans (three replicates ± SEM). [Figure 8A]Figures 8A-8D show confocal images of exemplary pig ear skin cross sections histologically stained after 24 hours of incubation with labeled Q-starch / HA with an N / O molar ratio of 0.25. Q-starch is labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch 5-DTAF), appearing as bright green staining in images 8A and 8C, and HA is labeled with Hylite™ Fluor 647 (HAHylite Fluor 647), appearing as red staining in images 8B and 8D. The nuclei of intact cells beneath the SC are stained blue (DAPI staining). Skin samples were either not pretreated with ultrasound prior to topical administration of the labeled complex Q-starch 5-DTAF-HAHylite Fluor 647 (8A, 8B) or were treated with US for 5 minutes prior to complex application (8C, 8D). Bar: 20 μm. [Figure 8B] Figures 8A-8D show confocal images of exemplary pig ear skin cross sections histologically stained after 24 hours of incubation with labeled Q-starch / HA with an N / O molar ratio of 0.25. Q-starch is labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch 5-DTAF), appearing as bright green staining in images 8A and 8C, and HA is labeled with Hylite™ Fluor 647 (HAHylite Fluor 647), appearing as red staining in images 8B and 8D. The nuclei of intact cells beneath the SC are stained blue (DAPI staining). Skin samples were either not pretreated with ultrasound prior to topical administration of the labeled complex Q-starch 5-DTAF-HAHylite Fluor 647 (8A, 8B) or were treated with US for 5 minutes prior to complex application (8C, 8D). Bar: 20 μm. [Figure 8C]Figures 8A-8D show confocal images of exemplary pig ear skin cross sections histologically stained after 24 hours of incubation with labeled Q-starch / HA with an N / O molar ratio of 0.25. Q-starch is labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch 5-DTAF), appearing as bright green staining in images 8A and 8C, and HA is labeled with Hylite™ Fluor 647 (HAHylite Fluor 647), appearing as red staining in images 8B and 8D. The nuclei of intact cells beneath the SC are stained blue (DAPI staining). Skin samples were either not pretreated with ultrasound prior to topical administration of the labeled complex Q-starch 5-DTAF-HAHylite Fluor 647 (8A, 8B) or were treated with US for 5 minutes prior to complex application (8C, 8D). Bar: 20 μm. [Figure 8D] Figures 8A-8D show confocal images of exemplary pig ear skin cross sections histologically stained after 24 hours of incubation with labeled Q-starch / HA with an N / O molar ratio of 0.25. Q-starch is labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch 5-DTAF), appearing as bright green staining in images 8A and 8C, and HA is labeled with Hylite™ Fluor 647 (HAHylite Fluor 647), appearing as red staining in images 8B and 8D. The nuclei of intact cells beneath the SC are stained blue (DAPI staining). Skin samples were either not pretreated with ultrasound prior to topical administration of the labeled complex Q-starch 5-DTAF-HAHylite Fluor 647 (8A, 8B) or were treated with US for 5 minutes prior to complex application (8C, 8D). Bar: 20 μm.

Claims

1. 1. A pharmaceutical composition comprising as an active ingredient hyaluronic acid (HA) complexed with a polysaccharide modified by substitution with one or more positively charged chemical moieties (HA-polysaccharide complex) for use in the non-invasive prevention or treatment of the skin aging process in a subject, The prevention or treatment includes: A pharmaceutical composition comprising applying ultrasonic treatment to the skin surface of said subject 5 seconds to 5 minutes prior to use of said pharmaceutical composition.

2. 10. The pharmaceutical composition of claim 1, wherein the application of ultrasonic treatment prior to use of the pharmaceutical composition is repeated at least once.

3. 2. The pharmaceutical composition of claim 1, wherein the molecular weight of the hyaluronic acid is less than 100 kDa, less than 500 kDa, more than 500 kDa, or within the range of 300 kDa to 800 kDa, 500 kDa to 1500 kDa, 500 kDa to 8000 kDa, 1000 kDa to 5000 kDa, or 500 kDa to 3000 kDa.

4. 2. The pharmaceutical composition of claim 1, wherein the prevention or treatment of the skin aging process is selected from the group consisting of maintaining skin hydration, restoring or improving collagen production, slowing the aging process, reducing aging indicators related to loss of mechanical properties, and slowing, reducing, or preventing at least one of wrinkles, skin atrophy, or loss of skin elasticity.

5. 5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the modified polysaccharides are starch, chitosan, pectin, cellulose, dextran, and galactan, and these polysaccharides optionally substituted with one or more quaternary amine groups.

6. 6. The pharmaceutical composition of claim 5, wherein the modified polysaccharide is a starch substituted with quaternary amine groups.

7. 7. The pharmaceutical composition of claim 6, wherein the quaternary amine-substituted starch is complexed with hyaluronic acid, and the molar ratio of the positively charged quaternary amine groups of the starch to the negatively charged carboxyl groups of the hyaluronic acid (N / O molar ratio) is within the range of 0.20-3.00, 0.22-0.50, 0.25-1.50, 1.00-2.50, or 0.

25.

8. 2. The pharmaceutical composition according to claim 1, wherein the duration of application of ultrasound is from 30 seconds to 2 minutes.

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