Methods for enhancing transdermal delivery of glycosaminoglycans (GAGs)

Abstract

The present invention provides compounds, compositions, and kits for promoting the delivery of high molecular weight hyaluronic acid to the deep layers of the epidermis and dermis. [Solution] A complex of hyaluronic acid and a chemically modified polysaccharide is provided. Preferably, the polysaccharide is modified by substitution with one or more positively charged chemical moieties, and more preferably, the positively charged chemical moieties are quaternary amine groups.

Description

Detailed description of the invention

[0001] [Field of Invention] This disclosure relates to, but is not limited to, a method for enhancing the transdermal delivery of glycosaminoglycans (GAGs), more specifically, to ultrasound-enhanced transdermal delivery of hyaluronic acid (HA).

[0002] 〔background〕 Human skin aging is a complex biological process mediated by a combination of two independent factors. The first process is endogenous or congenital aging, influenced by age-related hormonal changes such as a decrease in estrogen, androgens, and progesterone, which is associated with menopause and andropause (male menopause). Deficiencies in these hormones lead to collagen breakdown, dryness, loss of elasticity, skin atrophy, and wrinkles. The second process is extrinsic aging, which is a result of exposure to external factors, primarily ultraviolet (UV) irradiation. A key molecule involved in improving skin moisture and collagen production is hyaluronan or hyaluronic acid (HA), a glycosaminoglycan (GAG; a long, unbranched polysaccharide consisting of repeating disaccharide units) that forms the main component of the extracellular matrix (ECM). Young skin is hydrated because it contains a large 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 has decreased to 5 percent of the baseline. The combination of fibers and ECM provides the skin with viscoelastic properties and the resulting strength and elasticity. However, with age, tissue breakdown and degradation of dermal fibers and HA occur, leading to a decrease in the ability of HA to provide the skin with elasticity, density, and resistance.

[0003] The stratum corneum (SC), the outermost layer of the skin, provides mechanical protection to the skin and is a barrier against water loss and the penetration of substances from the environment. In particular, since the SC prevents the efficient penetration of large molecules (>500 Da), the effective utilization of HA with a large molecular size, which is administered topically, is limited due to skin permeability. In fact, topically applied HA does not completely penetrate the epidermis to the dermis. Therefore, when it is desirable to prevent or treat the skin aging process, HA is usually delivered to the deeper layers of the skin by injection. The main 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 into the blood vessels of the skin.

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

[0005] 〔Summary〕 When applied topically on 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. The delivery of high molecular weight HA to deeper skin layers is highly desirable as it reaches more tissues and has a longer duration. Currently, the transdermal delivery of high molecular weight HA for a wide range of applications is performed by intradermal (ID) injection of HA, i.e., an injection delivered into the dermis.

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

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

[0008] In one aspect, the present disclosure is a non-invasive method for preventing or treating the skin aging process in a subject that requires preventing or treating the skin aging process, comprising: (a) applying ultrasonic treatment to the skin surface of the subject for about 5 seconds to about 5 minutes; (b) topically administering at least one of free hyaluronic acid (HA) or HA complexed with a polysaccharide (HA-polysaccharide complex) to the ultrasonic-treated skin surface; and (c) optionally, repeating at least one of steps (a) or (b) at least once, thereby non-invasively preventing or treating the skin aging process in the subject.

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

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

[0011] In another aspect, the present disclosure is a method for the transdermal delivery of one or more glycosaminoglycans (GAGs) in a subject that requires the transdermal delivery of one or more glycosaminoglycans (GAGs), comprising: (a) optionally forming a complex (GAG-polysaccharide complex) comprising one or more GAGs and at least one polysaccharide; (b) A step of applying ultrasonic treatment to the target skin surface for approximately 5 seconds to approximately 5 minutes; (c) A step of topically administering one or more GAGs and / or one or more GAG-polysaccharide complexes to an ultrasonically treated skin surface; (d) optionally, a step of applying further ultrasonic treatment to the skin surface for about 5 seconds to about 5 minutes; and (e) optionally, a step of topically administering one or more GAGs and / or one or more GAG-polysaccharide complexes to an ultrasonically treated skin surface, This relates to a method for transdermally delivering one or more GAGs to a target.

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

[0013] In some embodiments, step (a) is applied, and at least one GAG-polysaccharide complex 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 method 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, for example, 500 kDa to 3000 kDa, or 300 kDa to 800 kDa.

[0016] The polysaccharide used in the intended method 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 a quaternary amine group (Q-starch) is used as a carrier for HA.

[0018] In a further embodiment, the present invention relates to a starch that is (CH3)3-N + This 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 complex is characterized by a molar ratio (N / O molar ratio) of the positively charged chemical moiety of the modified starch to the negatively charged carboxyl groups of hyaluronic acid, which is about 0.20 to about 3.00, for example, about 0.25 to about 1.5.

[0020] In further embodiments, the 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 composition to be considered may be a cosmetic composition or a therapeutic composition (i.e., a pharmaceutical).

[0021] In yet another embodiment, 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 the same; (b) means for applying sonication; 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 described herein may be used, for example, to enhance the non-invasive transdermal delivery of hyaluronic acid, preferably HMW HA, for the purpose of anti-aging therapy.

[0023] [Brief explanation of the drawing] Some embodiments of the present invention are described herein, merely as examples, in reference to the accompanying drawings. Hereinafter, with regard to the drawings in detail, it is emphasized that the details shown are provided as examples for the purpose of illustrative discussion of the embodiments described herein. In this regard, the description with reference to the drawings will make it clear to those skilled in the art how embodiments of the present disclosure may be carried out.

[0024] In the drawing: Figures 1A and 1B are graphs showing the size distribution of the quaternary starch (Q-starch) and hyaluronic acid (HA) complex (Q-starch-HA complex) (1A), as well as free Q-starch and HA (1B), obtained using dynamic light scattering (DLS). The Q-starch-HA complex is characterized by an increased ratio (N / O ratio) between the positively charged amine groups (N) of Q-starch and the negatively charged carboxyl groups (O) of the HA backbone; Figure 2 is a graph showing the size distribution (average diameter) of free Q-starch, free HA, and Q-starch-HA complexes with an N / O ratio of 0.25, as measured using the NanoSight system; Figure 3 is a bar graph showing the average zeta potential (action of particle surface charge) of free HA, Q-starch, and Q-starch-HA complexes, characterized by an increasing N / O ratio; Figures 4A–4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and newly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C–4G); Figures 5A and 5B show the results of Hylite® Fluor647 dye (HA) before (5A) ultrasound pretreatment, or after 5 minutes of ultrasound treatment (5B).Hylite Fluor 647 This is a bright-field confocal image of an exemplary pig ear skin cross section after 24-hour incubation with 0.3% (w / v) HA labeled with ). It shows the stratum corneum (SC), epidermis, and dermis (bar: 20 μm). HA was calculated for pixels in the exemplary rectangular cross section shown by image j as a function of distance from SC to a depth of 200 μm. Hylite Fluor 647 The fluorescence intensity is shown for each cross-section. Vertical dashed lines represent separation between layers. Horizontal dashed lines indicate the autofluorescence of the skin at the wavelength of labeled HA; Figures 6A to 6D show labeled Q-starch / HA complexes characterized by an N / O molar ratio of 0.25 (Q-starch-HA Hylite Fluor 647 These are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with Q-starch / HA. Skin samples were either not pre-treated with ultrasound before topical application of labeled Q-starch / HA complex (6A, 6B) or treated with 5 minutes of ultrasound before complex application (6C, 6D). Figures 6A and 6C are confocal images showing intact nucleated cells in the dermal layers 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). Hylite Fluor 647 The fluorescence intensity of Q-starch-HA was calculated as the distance from SC to a depth of 350 μm for pixels in the exemplary rectangular cross-section shown by image j. Hylite Fluor 647 The fluorescence intensity is shown for each cross-section. Vertical dashed lines represent separation between layers. Horizontal dashed lines indicate the autofluorescence of the skin at the wavelength of labeled HA; Figure 7 shows labeled HA in three layers of pig ear skin: SC (0-20 μm), epidermis (20-100 μm), and dermis (100-2000 μm). Hylite Fluor 647 This is a bar graph showing the fluorescence intensity measured at the wavelength of ). Three groups of skin samples were observed: (i) labeled Q-starch-HA complex (Q-starch-HA Hylite Fluor 647A skin sample to which (ii) was topically applied for 24 hours; a skin sample treated with ultrasound for 5 minutes and then Q-dextran-HA Hylite Fluor 647 A skin sample to which Hylite Fluor 647 was topically applied for 24 hours; and (iii) A skin sample that is not treated with either ultrasound or the labeled complex. This group serves for autofluorescence measurement. Fluorescence intensity was calculated by Image J based on data recorded from confocal scanning (3 replicates ± SEM); and Figures 8A - 8D are confocal images of a histological section of the skin of an exemplary pig ear stained after incubation with labeled Q-dextran / HA having a N / O molar ratio of 0.25 for 24 hours, where the Q-dextran is labeled with 5-(4,6-dichlorotriazinyl) aminofluorescein (5-DTAF) (Q-dextran 5-DTAF ), appears as bright green staining in Images 8A and 8C, the HA is labeled with Hylite™ Fluor 647 (HA Hylite Fluor 647 ), appears as red staining in Images 8B and 8D. The nuclei of the non-wounded cells under the SC are stained blue (DAPI staining). The skin samples were either not pretreated with ultrasound application prior to topical administration of the labeled complex Q-dextran 5-DTAF -HA Hylite Fluor 647 (8A, 8B) or treated with 5-minute US application prior to complex application (8C, 8D). Bar: 20 μm.

[0025] 〔Detailed Description〕 The present invention relates to, but is not limited to, a non-invasive means for enhancing the transdermal delivery of glycosaminoglycan (GAG), more specifically, the application of ultrasound for enhancing the transdermal delivery of hyaluronic acid (HA).

[0026] In the context of this disclosure, the term “transdermal delivery” should be interpreted broadly to include both (i) means of delivery of a substance through the skin, i.e., onto the skin (topically), for example, by application of a solution, ointment, patch, etc., to promote its systemic absorption; and (ii) delivery via the cutaneous layers of the upper outer stratum corneum (SC) to deeper cutaneous layers such as the epidermis and dermis, for example, 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 “intracutaneous delivery.” Accordingly, in any one of the embodiments described herein, transdermal delivery may apply to systemic delivery of GAGs through the skin and / or delivery of GAGs between cutaneous layers.

[0027] This disclosure is based on the inventors' discovery that the skin permeability of HA can be enhanced by applying ultrasound therapy to the skin. This disclosure is further based on the inventors' discovery that if HA is able to self-assemble with positively charged starch, i.e., starch substituted with positively charged moieties such as quaternary ammonium groups, an HA-starch complex is formed after ultrasound application to the skin that can be readily delivered transdermally, and furthermore, it is stable in the deeper layers of skin tissue. Such a complex gives HA higher intra-tissue stability compared to free acidic glycosaminoglycans, and therefore gives longer HA retention times in deeper layers of the skin, such as the epidermis and dermis.

[0028] The inventors envision therapeutic and cosmetic hyaluronic acid-based treatment modes in which HA is non-invasively injected percutaneously by utilizing the application of ultrasound and HA aggregates with quaternary starch (Q-starch). For example, the inventors envision combining ultrasound application with the Q-starch-HA complex in skin aging treatment modes such as wrinkle treatment, delay of the aging process, reduction of aging indicators associated with loss of mechanical properties, restoration of skin moisture, and skin smoothing. The combination of ultrasound application and HA complexation provides a convenient, non-invasive, and painless means of delivering HA to target skin layers such as the dermis, and further reduces the need for frequent percutaneous administration of HA.

[0029] Examples disclosed herein describe the successful insertion of high molecular weight hyaluronic acid (1500 kDa) primarily into the upper layers of the skin by applying ultrasound treatment to the skin prior to HA application. 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 for several molar ratios (referred herein to the N / O molar ratio or simply N / O) between the positively charged amine groups of Q-starch and the negatively charged carboxyl groups of HA. From skin penetration experiments disclosed herein, it is clearly seen that ultrasound application successfully provides greater introduction of Q-starch-HA complexes into deeper or lower layers of the skin, and uniform distribution of these complexes within them.

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

[0031] The epidermis, approximately 0.07 to 1.4 mm thick, is mainly composed of cells called "keratinocytes" and is organized into five layers representing different stages of cellular life in the epidermis. The layers are arranged from inside to outside as follows: (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 transparent cells with indistinct or absent nuclei. In the epidermis of the general body surface, the clear layer is usually absent, and (5) the horny layer (stratum The corneum (SC) is composed of hexagonal, flattened, keratinized, unnucleated cells called "keratinocytes," held together by lipids and desmosomes, commonly referred to as a brick-and-mortar structure. Desmosomes are specialized intercellular junctions formed by proteins, and together with lipids, maintain the integrity of the SC. The keratinocytes on 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, has a thickness of 10-15 μm. Upon hydration, the stratum corneum swells considerably, its thickness can reach up to 40 μm, accompanied by increased permeability.

[0032] The stratum corneum is the primary transport barrier for external substances. Furthermore, the stratum corneum 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 layer beneath the sclerotium divide to replenish their supplies. The epidermis does not undergo angiogenesis, and therefore, living keratinocytes in the epidermal layer obtain nutrients from the dermis, which is separated from the epidermis by the basement membrane (dermal-epidermal junction (DEJ)).

[0034] The dermis (true skin) is the fibrous inner layer of the skin directly beneath the epidermis, originating from the embryonic mesoderm, and ranging in thickness from 0.05 cm to 0.3 cm. The dermis contains fibroblasts, histiocytes, and mast cells, and 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 the skin with structure, elasticity, flexibility, and strength, protecting the body from mechanical damage. The dermis plays a vital role in maintaining epidermal properties, as well as repairing and restoring skin after injury.

[0035] The dermis consists of two regions: the epidermis (papillary layer, or dermal papillary layer), a thin surface layer interlocked with the epidermis, and a deeper, coarser reticular layer (or dermal reticular layer). 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 subcutaneous tissue (the innermost layer of the skin) and consists of thick collagen fibers that provide strength and elasticity to the skin, and contains hair follicles, sweat glands, and sebaceous glands.

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

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

[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 NH2 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, for example, the regulation of cell growth and proliferation, the promotion of cell adhesion, anticoagulation, and wound repair. The four main groups of GAGs are classified based on their core disaccharide units and include heparin / heparan sulfate, chondroitin sulfate / dermatan sulfate, keratan sulfate, and hyaluronic acid. The main categories of GAGs differ depending on the type of monosaccharide and the presence or absence of modification by sulfate.

[0039] In one embodiment, the present disclosure provides a non-invasive method for transdermal delivery of one or more glycosaminoglycans (GAGs) to subjects requiring transdermal delivery of one or more glycosaminoglycans (GAGs): (a) A step of applying ultrasonic treatment to the target skin surface for approximately 5 seconds to approximately 5 minutes; (b) The step of locally administering one or more GAGs to an ultrasonically treated skin surface; (c) Optionally, a step of applying further ultrasonic treatment to the skin surface for about 5 seconds to about 5 minutes; and (d) optionally, the step of topically administering one or more additional amounts of GAG to the sonicated skin surface, This relates to a method for non-invasive transdermal delivery of GAGs.

[0040] GAGs delivered transdermally during ultrasound pre-treatment, though not always, typically have a molecular weight of <500 kDa. Higher molecular weight GAGs, for example, GAGs with molecular weights in the range of 500 kDa to 8000 kDa, may be delivered according to this disclosure, but are not limited to, by aggregation or complexation with specific carrier polymers such as starch, chitosan, pectin, cellulose, dextran, or polysaccharides such as galactan, or optionally functionalized polysaccharides.

[0041] Such complexes have been previously utilized by the inventors as nonviral carriers of microRNAs (miRNAs) for the treatment of psoriasis, small interfering RNAs (siRNAs) for the treatment of ovarian cancer, and PI3P for the treatment of hepatic 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. Starch, in particular, 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 used in the intended method is functionalized starch.

[0043] Starch has the general formula (C6H 10 O5) nStarch is one of the polysaccharides and is the main storage form of carbohydrates in plants, and one of the natural energy stores. Starch is a biodegradable polymer composed of D-glucose residues consisting of 20% amylose and 80% amylopectin. Amylose contains α-1,4 links, and amylopectin further contains α-1,6 links. It is mainly found in the seeds, fruits, tubers, roots, and pith of plant stems, especially in maize, potatoes, wheat, and rice, and although its appearance varies greatly depending on the raw material, it is generally prepared as a white amorphous tasteless powder. Starch has many desirable characteristics, including low toxicity, biocompatibility, stability, low cost, hydrophilicity, and availability of reaction sites for chemical modification.

[0044] For starch to be an effective carrier of GAGs, since starch is an electrically neutral polysaccharide, it needs to undergo specific modifications (also referred to herein as “functionalization”), such as attaching a positively charged group to the starch. The term “quaternized starch” (Q-starch) refers to a starch molecule having a main chain that has undergone specific modifications, such as the substitution or addition of at least one quaternary moiety or quaternary group. A quaternary moiety or group is defined herein as a cation consisting of a positively charged atom at the center with four substituents. Such a cation is also referred herein as a “quaternary cation.” A “quaternary compound” as defined herein is a compound that is a quaternary cation or has a quaternary cation. The best known quaternary compound is a quaternary ammonium salt (N) with a positively charged nitrogen atom at the center. + R4 (where R is a substituent). Another example is a substituted phosphonium salt (R4P + ), and substituted arsoneum salts such as arsenobetaine (R4As + Examples include: For instance, quaternized potato starch can be obtained by substitution with a quaternary group, providing Q-starch with cationic properties. Q-starch can bind to molecules having negatively charged groups through the self-assembly of complexes.

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

[0046] In a further embodiment, this disclosure provides a non-invasive method for promoting the transdermal penetration of one or more GAGs having a molecular weight of 500 kDa to 8000 kDa into the deeper layers of the skin, i.e., into the deeper layers of the epidermis via the stratum corneum (SC), and, for example, into the dermis: (a) The process of applying ultrasound treatment to the target skin surface for approximately 5 seconds to approximately 5 minutes; (b) A step of topically administering one or more GAGs complexed with at least one polysaccharide to an ultrasonically treated skin surface; (c) Optionally, a step of applying further ultrasonic treatment to the skin surface for about 5 seconds to about 5 minutes; and (d) The present invention relates to a method comprising optionally administering a further amount of a GAG-polysaccharide complex topically to an ultrasonically treated skin surface, thereby promoting the penetration of GAG into the deeper layers of the skin.

[0047] Steps (c) and (d) may be repeated once, twice, three times, or more times, as necessary.

[0048] As used herein, “deep layers of skin” refers to the layers below the stratum corneum, such as the inner epidermal layers, including the stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale, and / or the dermis. “Deep layers of skin” also refers to the depths below the stratum corneum from 0 to approximately 2000 μm, for example, approximately 0 to approximately 10 μm, approximately 5 μm to approximately 20 μm, approximately 10 μm to approximately 30 μm, approximately 20 μm to approximately 40 μm, approximately 30 μm to approximately 60 μm, approximately 50 μm to approximately 80 μm, approximately 60 μm to approximately 100 μm, approximately 80 μm to approximately 120 μm, approximately 100 μm to approximately 150 μm, approximately 140 μm to approximately 200 μm, approximately 180 μm to approximately 250 μm, approximately 200 μm and below. This refers to approximately 270 μm, approximately 250 μm to approximately 300 μm, approximately 280 μm to approximately 350 μm, approximately 320 μm to approximately 400 μm, approximately 250 μm to approximately 500 μm, approximately 450 μm to approximately 600 μm, approximately 500 μm to approximately 800 μm, approximately 650 μm to approximately 900 μm, approximately 800 μm to approximately 1000 μm, approximately 900 μm to approximately 1500 μm, or approximately 1000 μm to approximately 1800 μm, as well as any sub-ranges and individual depths between them.

[0049] Glycosaminoglycans that can be delivered transdermally using the 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 treatment. 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 intercellular and cell-to-extracellular matrix adhesion. In malignant tumors such as melanoma, degradation of heparan sulfate in the extracellular matrix by the enzyme heparanase leads to the migration and metastasis of malignant cells. This mechanism makes heparanase and heparan sulfate viable pharmacological targets for the prevention of cancer metastasis.

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

[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 the articular cartilage of the joint, leading to inflammation and catabolism of the cartilage and subchondral bone. The structural modification role of chondroitin sulfate in OA is due to its role in stimulating the production of type II collagen and proteoglycans (PGs) in both the articular cartilage and synovial membrane. This anabolic effect of chondroitin sulfate prevents further tissue damage and remodeling of synovial tissue.

[0053] Keratan sulfate plays a functional role in both the cornea and the nervous system. The cornea contains the most abundant known source of keratan sulfate in the body, followed by brain tissue. The role of keratan sulfate in the cornea includes regulating the spacing of collagen fibrils, which is essential for optical clarity, as well as optimizing corneal hydration during development based on its interaction with water molecules. Like other GAGs, the degree of sulfated keratan sulfate determines its functional state. Keratan sulfate has also been shown to play a crucial regulatory role in the development of nervous tissue. Various subgroups of keratan sulfate in the brain play important roles in stimulating the proliferation of microglial cells and promoting axonal repair after injury.

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

[0055] Hyaluronic acid production is controlled by fibroblasts, keratinocytes, or chondrocytes. HA synthesis levels are very high in tissues such as skin and cartilage, where HA constitutes the majority of tissue masses. Hyaluronan has a dynamic metabolic turnover rate, with half-lives of 3-5 minutes in the blood, less than 1 day in the skin, and 1-3 weeks in cartilage. It is broken down into fragments of various sizes by hydrolysis, which occurs 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, in a process requiring the presence of molecular oxygen.

[0056] Hyaluronic acid is best known for its ability to attract water molecules. In physiological solutions, the carboxyl group of HA is negatively charged (anionic), allowing HA to form salts with mobile cations. These salts are highly hydrophilic and consequently surrounded by water molecules. The highly polar structure of HA allows it to bond up to 10,000 times its own weight in water. Water molecules bond to the HA carboxyl and acetamide groups via H bonds that stabilize the secondary structure of the biopolymer, described as a single-chain left-handed helix (2-turn helix) with two disaccharide residues per turn. In aqueous solution, these 2-turn helices form a double-chain, i.e., β-sheet tertiary structure due to hydrophobic interactions and intermolecular H bonds, which allows for the aggregation of polymer chains, resulting in the formation of an extended network structure. The HA network is strengthened as molecular weight (MW) and concentration increase. Due to these properties, HA plays a crucial role in synovial joint lubrication and wound healing processes.

[0057] Hyaluronic acid has a fairly wide molecular size / weight range (10 5 ~10 7 Hyaluronic acid (HMW HA) possesses hyaluronic acid (Da) and arises in a vast number of configurations and shapes depending on its size, salt concentration, pH, and associated cations. The biological function of HA is strongly dependent on its size: high molecular weight hyaluronic acid (HMW HA) chains (>5 × 10⁻¹⁰). 5Da) has space-filling, anti-angiogenic, immunosuppressive, cell proliferation inhibitory, and vascular endothelial cell mobility properties and is commonly used in various pharmaceutical applications (e.g., cancer treatment, osteoarthritis treatment, ophthalmic surgery, reconstructive surgery, drug delivery, and wound healing); less than 500 kDa (e.g., 2 × 10⁻¹⁰ 4 ~10 5 Medium-sized HA chains (between Da) are involved in ovulation, embryogenesis, and wound repair; less than 100kDa (e.g., 6×10) 3 ~2×10 4 Low molecular weight HA (LMW HA) chains (between Da) are inflammatory, immunostimulant, and angiogenic, while small HA oligomers (400-4000 Da) are anti-apoptotic and inducers of heat shock proteins. Low molecular weight HA and smaller oligosaccharides can be produced naturally in the body or artificially by controlling the depolymerization of HMW HA using physical treatments (heat treatment, pressure), irradiation, ultrasound application, acid treatment, radical oxidation, and enzymatic hydrolysis by hyaluronidase.

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

[0059] In cosmetics, HA shows promising efficacy in promoting skin tension and elasticity and improving aesthetic scores. For example, the use of HA in cosmetic formulations as a moisturizing active ingredient to restore the physiological microenvironment typical of young skin is well known and widely used. For cosmetic use, HA is classified by its molecular weight. Hyaluronic acid with a molecular weight of 20-300 kDa can penetrate the stratum corneum, and HA with a molecular weight of 5 kDa penetrates deeper into the epidermis, while HA with a higher molecular weight (500-1500 kDa) remains normally on the surface of the skin and cannot penetrate the stratum corneum (SC).

[0060] The hyaluronic acid used in the embodiments described herein includes any form of commercially available or custom-made HA produced by any technique known in the art, such as extraction from animal sources or microbial fermentation (e.g., fermentation of a strain of Streptococcus bacteria), but is not limited thereto.

[0061] Some embodiments relate to the use of chemically modified HA. Chemical modification of HA mainly involves two functional sites: hydroxyl (possibly the primary alcohol functional group of N-acetyl-D-glucosamine) and carboxyl groups. 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 an HA chain, each forming a single covalent bond, while crosslinking uses a polyfunctional compound that links different chains of natural or conjugated HA together by two or more covalent bonds. Crosslinked hyaluronanes can be prepared from natural HA (direct crosslinking) and / or HA conjugates (i.e., HA covalently bonded to one or more functional groups). In direct crosslinking of natural HA molecules, the hydroxyl and carboxyl groups can be crosslinked via ether and ester bonds, respectively. In some embodiments, HA is chemically modified before its crosslinking to introduce other chemically reactive groups. For example, HA may be treated with an acid or base to undergo at least partial deacetylation, resulting in the presence of a free amino group, which can then be crosslinked via an amide (-C(O)-NH-); imino (-N=CH-) or secondary amine (-NH-CH-) bond. The imino bond can be converted to an amine bond in the presence of a reducing agent.

[0062] Conjugation and crosslinking are generally performed for various purposes. For example, to obtain a carrier system with improved drug delivery properties or prodrug properties, conjugation provides crosslinking with various molecules. Crosslinking can further improve the mechanical, rheological, and swelling properties of HA, reduce its degradation rate, and provide HA derivatives with longer residence times and greater release properties at the application site. The higher the degree of crosslinking, the lower the water absorption capacity of the crosslinked HA and the higher its stability in aqueous solutions. Furthermore, double-crosslinked HA exhibits higher 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 any natural HA of any molecular weight known in the art, as well as any chemically or physically modified HA derivatives, including but not limited to HA conjugates and crosslinked HA.

[0064] Hyaluronic acid homeostasis changes with age, as well as due to external and internal treatments and drugs, such as sun exposure, which cause the breakdown of HMW HA. The HA content in the dermis is significantly higher than that in the epidermis. Both epidermal and dermal cells can synthesize HA throughout our lives. However, skin cells lose the ability to produce optimal amounts of HA during the aging process. The main 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 the binding and retention of water molecules, resulting in a loss of skin moisture. In the dermis, the main age-related change is an increase in the binding affinity (functional affinity) of HA to tissue structures, accompanied by a loss that occurs in conjunction with HA extractability. This is paralleled by the gradual cross-linking of collagen and the steady loss of collagen extractability with age. The decrease in HA production is also accompanied by a decrease in suppleness, elasticity, and skin tone loss, which characterize aging skin.

[0065] To maintain the aesthetic appearance of the skin and treat the signs of "dermatological" aging, it is recommended to continue "replenishing" the skin with HA from puberty onward. Today, available treatments for adding HA to the skin include serum, infusions, and oral ingestion. It is already known that orally ingested HA does not show any benefit to skin appearance, as skin cells cannot extract HA from the bloodstream. HMW Natural HA (>6×10 5 Topical application of HA (approximately 1 MDa) is difficult, mainly because its large size prevents efficient penetration into deeper skin layers. Instead, it forms a film that acts as a barrier against moisture loss. Furthermore, due to its good gelling properties, topical application of HA results in a hydration effect in the uppermost layer of the stratum corneum, and the accumulated water can swell and open the dense structure of the stratum corneum, leading to an increased degree of HA penetration into the upper layers of the epidermis. In this way, HMW HA has a favorable effect on the hydration of the upper epidermal layer, which manifests as lower transepidermal water loss. The skin's hydration capacity depends on the molecular size of HA, and for this reason, HMW HA (approximately 1 MDa) is usually added to cosmetic formulations.

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

[0067] In a further embodiment, the present disclosure relates to a non-invasive method for facilitating transdermal delivery of hyaluronic acid by applying ultrasound (US) treatment to the skin before topical application of HA. The method to be conceived comprises at least the following steps: (a) The process of applying ultrasound treatment to the target skin surface for approximately 5 seconds to approximately 5 minutes; and (b) A step of topically administering a hyaluronic acid solution to the ultrasonically treated skin surface; (c) Optionally, a step of applying further ultrasonic treatment to the skin surface for about 5 seconds to about 5 minutes; and (d) optionally includes the step of topically administering an additional amount of hyaluronic acid solution to the ultrasonically treated skin surface, This facilitates the transdermal delivery of hyaluronic acid to the target area.

[0068] In some embodiments, the intended method facilitates transdermal delivery of HA with a molecular weight in the range of 300–800 kDa to the epidermis, primarily the upper epidermal layer. 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 complex formation (e.g., assembling HA with a carrier such as a polysaccharide as described herein for transdermal delivery of GAGs) to further increase the skin penetration of MW into HA and its penetration into deeper layers of the epidermis.

[0070] Embodiments of the present disclosure are non-invasive methods for enabling or facilitating the penetration of high MW hyaluronic acid into the deeper layers of the skin of subjects requiring the penetration of high MW hyaluronic acid into the deeper layers of the skin: (a) A step of applying ultrasonic treatment to the target skin surface for approximately 5 seconds to approximately 5 minutes; (b) The present invention relates to a method comprising the step of topically administering a complex comprising hyaluronic acid and polysaccharides to an ultrasonically treated skin surface, thereby promoting the penetration of hyaluronic acid into the deeper layers of the skin in question.

[0071] This method is beneficial for transdermal delivery (i.e., delivery by skin and / or intradermal delivery) of HA with molecular weights >500kDa, for example, 500kDa to 8000kDa, 1000kDa to 5000kDa, or 500kDa to 3000kDa.

[0072] Optionally, steps (a) and (b) of the disclosed method may be repeated at least once (e.g., once, twice, three times, or more) as necessary to achieve efficient penetration of hyaluronic acid into the deeper layers of the skin; that is, after the initial ultrasound application, further ultrasound treatment may be applied to the treated skin surface for about 5 seconds to about 5 minutes, after which an additional amount of hyaluronic acid-polysaccharide complex may be optionally administered topically. In some embodiments, no additional HA administration is performed simultaneously after a second or third ultrasound application to the treated skin.

[0073] In some embodiments, non-invasive methods intended to facilitate the transdermal delivery of high-molecular-weight or low-molecular-weight hyaluronic acid to the deeper layers of the skin are utilized to treat or prevent skin aging processes in subjects requiring such treatment.

[0074] "Preventing or treating the skin aging process" as used herein means at least one of the following: maintaining skin hydration, restoring or improving collagen production, delaying aging processes such as wrinkles, or reducing aging indicators associated with loss of elasticity and loss of mechanical properties such as skin atrophy.

[0075] Skin atrophy is a common symptom of aging and is often accompanied by ulcer formation and delayed wound healing. Atrophic skin exhibits reduced HA content and expression of the main cell surface hyaluronic acid receptor, CD44. As the patient population ages, the management of skin atrophy is becoming a major challenge in clinics, especially given 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 a quaternary amine that forms a nano-sized complex (also referred to herein as a “nanocomplex”) with HA.

[0077] Ultrasound (referred to as "US" in this specification for brevity) is a sound wave with a frequency above 18 kHz, which is the limit of human hearing. Ultrasound is a longitudinal wave, meaning that 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 a medium, resulting in pressure fluctuations in the medium. The ultrasonic frequency (f) is the number of pressure fluctuation cycles in the medium per unit time (vibration velocity), measured in Hertz (Hz), where each cycle consists of compression and dilution. The amplitude (A) of the wave represents the maximum local pressure, measured in Pascals (Pa).

[0078] A typical ultrasonic induction device includes a piezoelectric transducer that converts an electrical signal into ultrasound. By applying an AC voltage to a piezoelectric material, the material vibrates at the same frequency as the driving current. The transducer can operate in continuous mode (repetitive cycles) or pulsed mode (temporally separated cycles with gaps without a signal).

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

[0080] The effects of ultrasound on biological tissues primarily include thermal heating, acoustic cavitation, and acoustic streaming. Thermal heating is a result of ultrasound waves passing through a medium. The sound waves of ultrasound are absorbed by the medium, inducing the formation of heat that can be conducted, convected, or radiated. The thermal effect increases with frequency, being most pronounced at megahertz frequencies. Low-frequency ultrasound has demonstrated the ability to significantly increase skin permeability, allowing the delivery of various substances through the skin. The main mechanism explaining the ability of ultrasound to increase skin permeability is acoustic cavitation, which can instantaneously induce the growth and vibration of air pockets present in 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 in areas of relatively low pressure. Expansion cycles in a medium create negative pressure, pulling molecules apart. When the pressure amplitude exceeds the tensile strength of the liquid in the dilution region, small vapor-filled cavities are formed. When exposed to higher pressures, these cavities, also called "cavitation bubbles" or "voids," collapse, potentially generating strong shock waves. Cavitation in liquid media, i.e., the formation of gas cavities, can be a 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 sound waves.

[0082] The term “acoustic cavitation,” as used herein, refers to the formation of bubbles in a medium exposed to ultrasound, and the activation (growth, vibration, or collapse) of existing bubbles. When existing bubbles in a liquid medium are exposed to ultrasound, they vibrate or collapse, producing 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 called “transient cavitation”). Stable cavitation is the long-duration vibration (a considerable number of cycles) of a bubble in response to a pressure change. The bubble expands during the dilution phase and contracts during the compression phase, vibrating around an equilibrium radius for several cycles. Stable vibration creates a flow of liquid around the bubble, known as microstreaming, which induces shear stress. If the bubble is 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 (inertial cavitation threshold). Bubbles grow violently and collapse; during collapse, symmetrical shock waves with high pressure (above 10 kbar) and temperature may be generated in the vicinity of the collapsing bubble. If the bubble is close to a solid surface, the collapse is asymmetric and a liquid jet may be generated. If the collapse occurs near biological tissue, it can lead to membrane perforation, reversible pore formation, and / or vascular permeability.

[0084] Acoustic cavitation is utilized in embodiments described herein to increase skin permeability, resulting in transdermal delivery of various GAGs through the skin (for systemic delivery) and between skin layers (for intradermal delivery). While not intended to be limited by theory, it is hypothesized that cavitation causes disorder in the lipids of the stratum corneum, and in areas of disordered lipids, water penetrates and promotes the formation of aqua channels. These channels in the intercellular lipids of SCs enable the transport of large molecules. Three modes of cavitation effect induced by US are hypothesized: shock waves, collision of microjet into SCs, and penetration of microjet into SCs. Both microjet and shock wave therapy may be responsible for the SC permeability-enhancing effect, with microjet being significantly more effective in increasing skin permeability (see, for example, 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, the suppositories (US) are surfactants ranging from hydrophobic agents such as oleic acid to hydrophilic sodium lauryl sulfate (SLS), but are not limited to these, and are applied in combination with the simultaneous topical application of one or more skin penetration enhancers. Surfactants are found in many existing therapeutic, cosmetic, and pesticide preparations and have recently been used to increase the permeability of several drugs via transdermal pathways. Surfactants affect the permeability properties of several biological membranes, including the skin. They have the potential to solubilize lipids in the stratum corneum. The penetration of surfactant molecules into the lipid lamellae of the stratum corneum is strongly dependent on the distribution behavior and solubility of the surfactant.

[0086] The inventors have determined that US (for example, 3 W / cm²) 2It was found that simultaneous application of US (0.5s on and 0.5s off) and SLS (1% solution) resulted in a change in the pH of SC, which affected both the structure of the lipid layer and the solubility of SLS within 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 generally increase skin permeability to GAGs and especially HAs.

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

[0089] (A complex of hyaluronic acid and modified starch) Aspects of this disclosure relate to a complex of hyaluronic acid with chemically modified starch. Such a complex acts as a carrier that facilitates the delivery of hyaluronic acid to the deeper layers of the skin, as defined herein. The disclosed complex is particularly useful for delivering high molecular weight hyaluronic acid.

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

[0091] The composites referred to herein include products such as conjugation, self-assembly, and crosslinking between HA and a carrier polymer, as well as encapsulation of HA by the carrier polymer. The composites intended herein are typically nanoscale in size and are also referred to herein as nanocomposites or nanocarriers.

[0092] The embodiments described herein relate to hyaluronic acid complexes having quaternized starch (Q-starch) obtained by the self-assembly of two polymers. The modified starch is obtained by covalently bonding (i.e., substituting) at least one quaternary amine group to it. Q-starch can be obtained by reaction 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 + - is

[0094] A complex of Q-starch and hyaluronic acid (hereinafter referred to as "Q-starch-HA") is designed and manufactured to feature a desired molar ratio (hereinafter referred to as the "N / O molar ratio," "N / O ratio," or simply "N / O") between the positively charged chemical moiety of the modified starch and the negatively charged carboxyl groups of hyaluronic acid. The N / O molar ratio affects complex formation and / or transdermal penetration effects. Possible Q-starch-HAs may feature an N / O ratio in the range of about 0.20 to about 3.50, among other things, depending on the molecular weight of hyaluronic acid and / or the type and MW of Q-starch. For example, N / O is approximately 0.20-0.40, 0.22-0.26, 0.25-0.35, 0.30-0.45, 0.40-0.60, 0.50-0.70, 0.65-0.80, 0.75-0.90, 0.80-1.00, 0.85-1.10, and 1.00-1. 20, any ratio in the range of approximately 1.10 to 1.40, approximately 1.25 to 1.50, approximately 1.35 to 1.65, approximately 1.50 to 1.85, approximately 1.70 to 2.00, approximately 2.10 to 2.50, approximately 2.30 to 2.65, or approximately 2.60 to 3.00, and any subranges and individual values ​​between them.

[0095] In some embodiments, N / O is in the range of about 0.22 to about 0.50, about 0.25 to about 1.50, or about 1.00 to about 2.50. In some embodiments, N / O is 0.25.

[0096] (Pharmaceutical composition) In further embodiments, this disclosure relates to compositions comprising one or more Q-starch-HA complexes described herein and physiologically acceptable excipients. The disclosed compositions may be cosmetic compositions and / or pharmaceutical or therapeutic compositions and may have therapeutic properties. In some embodiments, the compositions are formulated for transdermal administration and include physiologically acceptable carriers.

[0097] The terms “pharmaceutical composition” and “cosmetic composition,” as used herein, refer to a composition essentially comprising at least one Q-starch-HA complex, which may be employed for clinical or cosmetic use (but not limited to therapeutic or anti-aging uses, respectively). “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 applicable to administration to a subject. Such a formulation is referred herein to as a “Q-starch-HA formulation.” For example, a Q-starch-HA formulation may be formulated for topical or transdermal administration and may contain, for example, one or more Q-starch-HA complexes combined with or formulated together with one or more carriers, excipients, penetration enhancers, stabilizers, etc.

[0098] As used herein, the terms “pharmaceutically acceptable,” “pharmacologically acceptable,” and “physiologically acceptable” are interchangeable and mean approved by federal or state regulatory authorities, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals, more specifically in humans. These terms include, where applicable, formulations, molecular entities, excipients, carriers, and compositions that, when administered to animals or humans, do not produce adverse, allergic, or other undesirable reactions. For administration to humans, preparations should meet sterility, pyrogenicity, general safety, and purity standards, such as those required by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA).

[0099] In this specification, the term “excipient” refers to an inert substance added to a pharmaceutical composition or pharmaceutical formulation to further facilitate the processing and administration of the active ingredient. “Pharmacologically acceptable excipients,” as used herein, include approved preservatives, antioxidants, surfactants (e.g., Tween®-20, Tween®-40, Tween®-60, and Tween®-80), buffers, coatings, isotonic agents, absorption retarders, penetration enhancers, carriers, etc., that are suitable for pharmaceutically acceptable administration, do not cause significant irritation to the organism, and do not negate the biological activity and properties of the possible activator. Physiologically suitable carriers in liquid formulations may be, for example, solvents or dispersion media.

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

[0101] The intended kit is useful for enhancing the non-invasive transdermal delivery of hyaluronic acid, such as high molecular weight (>1000kDa) hyaluronic acid, particularly to the deep layers of the epidermis and / or dermis, which may find use in anti-aging treatments as well as any other therapeutic modes utilizing hyaluronic acid.

[0102] For clarity, it should be understood that certain features of the Disclosure described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the Disclosure described in the context of a single embodiment may be provided separately, in any suitable subcombination, or as appropriate in any other described embodiment of the Disclosure. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiments would not function without those elements.

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

[0104] The terms "comprises," "comprising," "includes," "including," "possess," and their conjugates all mean "includes but not limited to."

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

[0106] Throughout this description, various embodiments may be presented in range form. It should be understood that range form descriptions are merely for convenience and brevity and should not be interpreted as inflexible limitations on the scope of this disclosure. Therefore, range descriptions should be understood to include all specifically disclosed subranges, as well as the individual numbers within those ranges. For example, a range description such as 1–6 should be understood to include specifically disclosed subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, as well as the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0107] Various embodiments and aspects of the present invention, as described above in this specification and as set forth in the following claims, find experimental support in the following examples.

[0108] [Examples] The following examples, along with the above description, non-limitingly illustrate some embodiments of the present disclosure. Generally, the nomenclature used herein and the experimental procedures utilized herein include molecular, chemical, biochemical, and / or microbiological techniques. Such techniques are well described in the literature. Other general references are provided throughout this specification, and the procedures therein are considered 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.: sodium N-hydroxysulfosuccinimide (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 Life core Biomedical. Soluble starch (101252) was purchased from Merck. We purchased Prolong gold colorfastness inhibitor (P36935) containing DAPI from Invitrogen. We purchased full-thickness skin derived from pig ears from the Institute of Animal Research, Lahav, Israel. We purchased Hylite® Fluor647nm amine (81257) from Anaspec.

[0110] (i) Hyaluronic acid labeling Labeling of HA was carried out as previously described by Sapir et al. (Biomaterials, 2011, 32.7: 1838-1847). The fluorescently labeled dye covalently bonded to HA via carbodiimide chemistry, creating an amide bond between the terminal amine group of the fluorescent molecule and the carboxyl group on HA. Briefly, 10 ml of 0.2% (w / v) aqueous solution of HA was prepared, and 426 mg of 4-morpholine ethanesulfonic acid monohydrate (MES-H2O) and 200 mg of NaCl were added to obtain a pH 6.5 solution. The mixture was stirred at room temperature for 10 minutes. The carboxyl groups on HA were activated by adding 38.4 mg / 0.5 ml of 1-ethyl(dimethylaminopropyl)-carbodiimide (EDAC) in double-distilled water (DDW), and the reactive intermediate was stabilized by adding 21.6 mg / 0.5 ml of the co-reactant N-hydroxysulfosuccinimide (sulfo-NHS) in DDW. The mixture was stirred at room temperature for 3 hours, and 1 mg / 0.5 ml of Hylite® Fluor647 amine dye in DDW was added. Stirring was continued for a further 12 hours to ensure the formation of amide bonds between the amine groups of the dye and the carboxyl groups of HA. The synthesized product (labeled HA) was purified by dialysis bag at a molecular weight cutoff of 11 kDa (MWCO) and placed in a container with 5 L of distilled water (DW). During dialysis over 3 days, the water was replaced 6 times with fresh DW. The dialyzed product was then freeze-dried for 72 hours and stored dry at 4°C.

[0111] (ii) Quaternary starch The modification or derivatization of starch by converting it into a cationic polymer is essential for enabling self-assembly with hyaluronic acid via 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] The 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 outlined in Scheme 1 below, as previously described by Amar-Lewis et al. (Journal of controlled release 185:109-120, 2014). First, 500 mg of water-soluble potato starch (hydrolyzed potato starch, MW26,765Da) 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. 9 grams of the quaternization 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 a starch solution. The reaction mixture was continuously stirred at room temperature for 24 hours. To precipitate the product, 1 volume of the 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 in a container with 5 L of DW. Dialysis was performed to remove unreacted cationic reagents. During 48 hours of dialysis, the water was replaced four times with fresh DW. The dialyzed product was then freeze-dried for 72 hours.

[0113] Scheme 1 shows the quaternization reaction of starch using the quaternization reagent CHMAC:

[0114] [ka]

[0115] The quaternization of starch was confirmed by Fourier transform infrared spectroscopy (FT-IR) and elemental analysis (EA). Measurements were obtained using a Thermo Nicolet® FT-IR spectrophotometer (Nicolet® iS® 10FT-IR spectrophotometer). 3027cm² -1and 1478cm -1 The observation of Q-starch was confirmed by the strong, novel absorption bands that appeared. These bands are associated with the stretching vibrations of the CH and CN in the quaternary ammonium group (CH3)3N-, respectively. The remaining bands were similar when compared to the FT-IR spectrum of unmodified starch. The sample was 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 positively charged amine groups (N) on Q-starch to negatively charged carboxyl groups (O) on the HA backbone is based on the amount of positive amine groups per starch chain. The weight percentage (N%) of nitrogen atoms in 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%. Based on calculations based on quaternization at the 6' position in each glucose monomer of starch, 4.2% is considered to be the maximum substitution. The weight percentage of nitrogen in Q-starch is calculated using Equation 1:

[0117]

number

[0118] The most significant advantage of using quaternary amines as substituted molecules is the polymer's charge, which is largely independent of the solution's pH. Unlike chitosan complexes, which are highly pH-dependent and remain stable primarily under acidic conditions, quaternized starch retains its positive charge over a wide pH range.

[0119] (iii) Q starch label 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, adjusting the pH 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 in the dark at room temperature for 24 hours. 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 in PBS buffer (pH 7.5) for 72 hours, followed by 48 hours in 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 composites 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-TEM to measure the size and shape of the composite.

[0121] (a) Zeta potential (ζ potential) The zeta potential (ζ potential) is the electric charge that arises at the interface between a solid surface and its liquid medium. Measuring in millivolts, this potential can arise from one of several mechanisms, including the dissociation of ionoid groups on the particle surface and differential adsorption of solution ions to the surface region. The net charge on the particle surface affects the ion distribution in nearby regions, increasing the concentration of counterions near the surface. Thus, an electric double layer is formed in the particle-liquid interface region, consisting of two parts: an inner region containing ions relatively densely bound to the surface, and an outer region where the balance between electrostatic force and random thermal motion determines the ion distribution. Consequently, the potential in this region decays with increasing distance from the surface, reaching the bulk solution value at a sufficient distance, and has conventionally been considered zero.

[0122] In an electric field, each particle and its most closely related ions move as a unit through the solution, and the potential at the shear surface 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 present on the hydrodynamic surface of the shear. When a layer of macromolecules is adsorbed onto the surface of a particle, it further shifts the shear surface away from the surface and alters the zeta potential.

[0123] Measuring zeta potential is currently the simplest and easiest method for characterizing the surface of charged colloids and is most relevant to the practical study and control of colloidal stability and aggregation processes.

[0124] The surface charges of hyaluronic acid, Q-starch, and Q-starch-HA complexes were measured by zeta potential measurement using a Zetasizer (ZN-NanoSizer, Malvern, England). The complexes were prepared with different N / O ratios (e.g., 0.25-0.3) as described in Example 1 below, and diluted to obtain a final HA concentration of 26 mM in a volume of 1 ml of DDW. The samples were transferred to a U-tube cuvette (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 is shown as the average of three runs (3 ± standard deviation).

[0125] (b) Dynamic light scattering (DLS) Dynamic light scattering (DLS) measures the temporal variation of light scattered by the Brownian motion of particles 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 relation to its hydrodynamic diameter. DLS is a highly sensitive, non-invasive, and powerful analytical tool routinely used for characterizing polymers, colloids, and nanoparticles in solution.

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

[0127] (c) Nanosite Nanosite analysis is a technique that allows for the sizing and quantification of nanoparticles through the use of light scattering. Unlike conventional DLS, it tracks the movement of individual nanoparticles in real time using a charge-coupled device (CCD) camera, and the system instrument derives their hydrodynamic radii through the Stokes-Einstein equations. Nanosite also surpasses DLS in that it can explicitly quantify particles smaller than 1 micron, allowing for more accurate characterization of polydisperse samples. When irradiated with laser light, the Nanosite nanoparticle analysis instrument generates a video of a collection of nanoparticles moving under Brownian motion in a liquid. Within a specially designed and constructed laser irradiation device mounted beneath the microscope objective lens, particles in the liquid sample passing through the beam path are seen by the instrument as tiny points of light rapidly moving under Brownian motion. This ability of the Nanosite system allows for dynamic analysis of the path that particles take under Brownian motion over a suitable time (e.g., 30 seconds).

[0128] The diameter of the Q-starch / HA composite in aqueous solution was determined using nanosite technique. The composite was prepared with an N / O molar ratio of 0.25–3 as described in Example 1 herein and diluted to a final concentration of 13 mM with a final volume of 2 ml of DDW. A nanosite-range NS300 instrument (Malvern Instruments, Malvern, UK) 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 lens, and 60-second video clips were recorded.

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

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

[0131] The size and shape of HA, Q-starch, and Q-starch-HA composites in solution were visualized and characterized by direct imaging of the aqueous solutions using cryo-TEM. As described in Example 1, the composites were prepared in 40 μl of DDW with a final HA concentration of 260 mM at a N / O ratio of 0.25. A drop of 2.5 μl of the solution was placed on a carbon lacy film supported on a 300-mesh Cu grid (PELCO® TEM, Ted Pella Ltd). Excess liquid was blotted, and the sample was vitrified by rapidly immersion in liquid ethane pre-cooled with liquid nitrogen in a controlled environment automated vitrification system (Leica EM GP) with controlled temperature and relative moisture. The sample was operated at 120 kV and on an FEI Tecnai® G with a Gatan626 cold stage control unit. 2 The samples were examined at -178°C using a 12-TWIN transmission electron microscope. 2D images were captured using a Gatan794MultiScan charge-coupled device (CCD) camera.

[0132] (v) Skin treatment For in vitro experiments, full-thickness skin derived from pig ears (from the back of the ear) was used. The skin was separated from the ear using a surgical scalpel, cut into 2x2 cm pieces, and frozen (-20°C) until use. Before each experiment, the skin samples were thawed to room temperature for 10 minutes.

[0133] (vi) Skin conductivity measurement Skin integrity and the effectiveness of ultrasonic pretreatment were evaluated by measuring skin conductivity. Ag / AgCl 4mm disk electrodes were introduced into both diffusion cell compartments in the in vitro experiment. An AC voltage of 200mV at 10Hz 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) Method and apparatus for measuring skin permeability in vitro Skin permeability measurements were performed in a vertical static glass diffusion cell consisting of a donor compartment and a receiver compartment. Skin was positioned between the two separate compartments so that the stratum corneum (SC) faced 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. Ultrasound-treated skin samples were subjected to ultrasound (QSonica Q700 Sonicator, frequency = 20 kHz, 8.2 W / cm²). 2 (3% amplitude, probe diameter 1.3 cm) was applied as a pretreatment: the ultrasound probe was placed 8 mm above the skin surface in the donor compartment. 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 culture medium at room temperature 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 start of the experiment, before and during US exposure. Conductivity was 0.7 (kΩ*cm). 2 ) -1Skin samples with higher conductivity were considered defective and were not used. The US was turned off 5 minutes after exposure, during which time all skin samples reached conductivity 50-60 times higher than their initial conductivity.

[0135] After US pretreatment, the skin is removed from the diffusion cell, washed with PBS, and returned to the cell with 700 μl of fluorescently labeled HA (HA) at the desired N / O molar ratio (e.g., 0.25) and HA concentration of 260 mM. 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 The sample was placed on the skin for 24 hours, then fixed in 4% paraformaldehyde and embedded in paraffin. After deparaffinization, 5 μm thick sections were cut from each sample, 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 on an LSM-880 using a ZEISS (Germany) Airyscan confocal system with a plan-apochromat 20× / 0.8DIC M27 objective lens.

[0136] To visualize the autofluorescence of skin, excitation was performed with a 488 nm argon laser, and emission was detected in the rage range of 490 nm to 597 nm. To visualize HA labeled with Hylite™ Fluor647, excitation was performed with a 633 nm HeNe laser, and emission was detected in the rage range of 638 nm 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 enhancement of fluorescence when bound to the AT region of dsDNA. It is excited by a violet (405 nm) laser beam. Because DAPI can pass through intact cell membranes, it can be used to stain both live and fixed cells. For DAPI staining, skin tissue was fixed in 4% formalin and embedded in paraffin wax. After deparaffinization, sections were made 5 μm thick using a microtome (Leica RM2255 microtome, England) and rehydrated. Before staining, slides were first washed with xylene (twice each for 10 minutes), then with 100% ethanol (twice each for 10 minutes), 95% ethanol (5 minutes), 70% ethanol (5 minutes), 50% ethanol (5 minutes), distilled water (5 minutes), and PBS (twice for 10 minutes). After cleaning, the slides were mounted using DAPI for nucleus visualization.

[0138] (Example 1) (Formation and characterization of the Q-starch-HA complex) Q-starch-HA complexes were prepared with different molar ratios (N / O molar ratio) between the positively charged amine groups (N) of Q-starch and the negatively charged carboxyl groups (O) of the HA backbone. For each measurement, the amount of HA (X mg of HA) was predetermined, and the amount of O (moles of O) in the carboxyl groups on HA was calculated according to Equation 2:

[0139]

number

[0140] The amount of Q-starch required for the desired N / O ratio 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 %) of N using Equation 3. Desired amounts of Q-starch and HA solutions were taken to prepare the complex, gently vortexed, and then incubated at room temperature for 40 minutes to allow for complex formation by self-assembly. The Q-starch-HA complex was prepared in an Eppendorf flask so that the amount of HA remained 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 the complex solution was 260 mM HA.

[0143] The size, surface charge, and morphology of Q-starch / HA composites are crucial parameters, as their values ​​can influence their penetration into the skin. The physical properties of Q-starch-HA composites at different N / O ratios were obtained using zeta potential for measuring surface charge, nanosites for diameter size, dynamic light scattering (DLS) for hydrodynamic sizing, and cryo-TEM for measuring the size and shape of the composites, as described in Materials and Methods.

[0144] (i) Characterization of the composite by dynamic light scattering (DLS) and nanosites Dynamic light scattering (DLS) and nanosite techniques were used to determine the average hydrodynamic radius and diameter of Q-starch / HA composites. Using dynamic light scattering, scattered light interacting with the composites suspended in the medium was measured, and its diffusion coefficient was calculated. Their hydrodynamic radii were then calculated using the Stokes-Einstein equation. Figure 1A shows the size distribution of Q-starch-HA composites at increasing N / O ratios. As observed, 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 distribution of the composites for each N / O ratio was relatively uniform, with all peaks 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, in contrast to composites showing narrow size distributions, clearly indicate the lack of evidence for internal self-interactions to form particles.

[0145] To validate 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. Both the plots for free HA and free Q-starch show a very wide range of size distributions with many indistinct peaks, but the plot for the complex shows a relatively narrow size distribution. When measured by nanosite, no significant difference was observed in the size distribution (mean diameter) of the Q-starch / HA complex with an increasing N / O ratio (results not shown), which is consistent with the results obtained from the DLS measurements.

[0146] (ii) Characterization of the zeta potential complex The zeta potential is the effect of the surface charge of the particles, any adsorbed layers at the interface, and the properties and composition of the surrounding suspension medium. To determine the surface charge of the composites at different N / O molar ratios, zeta potential measurements were performed on Q-starch-HA composites with different N / O ratios, as well as on free HA and newly prepared uncomposited 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 (-70mV) for uncomposited HA and a very positive zeta potential of 42mV for uncomposited 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 from a negative value of approximately -36mV for N / O 0.25 to a positive value of approximately 40mV for N / O 3.

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

[0148] The time-dependent stability of Q-starch-HA composites in aqueous solutions was evaluated by assessing their hydrodynamic size 3, 24, and 48 hours after their formation. For example, composites characterized by N / O 0.25 were evaluated by nanosite and cryo-TEM, and while their size increased slightly over time (presumably due to HA swelling in the aqueous medium), their diameter did not double or increase significantly, clearly indicating that no aggregates of the composites formed, i.e., the composites were stable (results not shown).

[0149] (Example 2) (Effects of ultrasound irradiation on the skin permeability of hyaluronic acid solution) In vitro uptake of fluorescently labeled high molecular weight (HMW) HA solution by porcine skin samples was measured with and 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. As described in Materials and Methods, the HA was fluorescently labeled with Hylite® Fluor647 amine dye (referred to herein as HA). Hylite Fluor647 (referred to as) Before providing the labeled HA to the skin sample, apply US for 5 minutes (20KHz, 8.2W / cm²) as described in Materials and Methods. 2 A duty cycle of 50% was used as a pretreatment. Exemplary confocal microscopy images of pig ear skin sections 24 hours after US application are shown in Figures 5A to 5C. The respective graphs show the fluorescence intensity of labeled HA as a function of distance from SC to a depth of 200 μm, calculated for pixels in an exemplary rectangular cross-section from image j.

[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 skin samples pretreated with US, the fluorescence intensity was higher in the epidermis and scattered to a depth of 50 μm in the layer below the SC layer (Figure 5B). These results confirm that US application can influence skin penetration beyond the SC layer.

[0151] One possible explanation for this phenomenon of increased SC permeability is a mechanical effect caused by US application such as cavitation. As discussed herein, both microjet and shock waves can contribute to the increase in SC permeability.

[0152] However, HA hardly penetrated to the dermis, the target layer of HA's biological activity. This can be explained by the fact that HMW HA is a large molecule, which slows its diffusion through the skin, especially in aqueous solutions.

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

[0154] The penetration of the Q-starch-HA complex into the deep skin layer 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® Fluor647 amine dye (HA). Hylite Fluor647 Labeled with ), the complex, Q-starch-HA Hylite Fluor647The compound was formed with an N / O molar ratio of 0.25 and applied topically to skin samples for 24 hours with or without ultrasound pre-treatment (5 minutes). Untreated (no ultrasound treatment and no complex administration) ear skin sections were used as a control group. This control group was used to evaluate autofluorescence at various depths or layers of skin samples. Visualization of histologically stained treated and untreated (control) pig skin sections was performed using a confocal microscope (excitation was performed with a 633 nm HeNe laser, and emission was detected at 638 nm to 759 nm), and exemplary confocal and bright-field images of treated skin are shown in Figures 6A to 6D. Complexed HA in DAPI-stained sections. Hylite Fluor647 The HA appeared as a red stain, and the cell nucleus was stained blue. In bright-field confocal images, the complexed HA was visible. Hylite Fluor647 It appeared as a pinkish-red color. Q-starch-HA Hylite Fluor647 The fluorescence intensity of the complex is Q-starch-HA as a function of the distance from SC to a depth of 350 μm, calculated (by image j), per pixel of any skin cross-section indicated by rectangles in the image. Hylite Fluor647 The fluorescence intensity of the complex is presented for both US-pretreated and untreated skin. For convenience, in this specification, the fluorescence intensity calculated for any pixel is referred to as “pixel fluorescence intensity”.

[0155] As seen in Figures 6A and 6B, in skin that was not pretreated with US before application of the complexed HA, the topically administered complex remained mostly in the SC layer, the topmost layer of skin. As calculated, the pixel fluorescence intensity was higher in the SC layer than in the epidermis and dermis (although some fluorescence was detected in these deeper layers as well). In contrast, Q-starch-HA Hylite Fluor647 In skin samples pre-treated with US for 5 minutes before topical application of the complex for 24 hours, 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 the Q-starch / HA complex in the deep skin layer between US-pretreated and unpretreated skin groups, skin samples from three groups were observed: (i) Labeled Q-starch-HA complex (Q-starch-HA Hylite Fluor647 (ii) Skin samples to which ) has been applied locally for 24 hours; (ii) Skin samples treated with ultrasound for 5 minutes, then Q-starch-HA Hylite Fluor647 (iii) Skin samples to which the product was applied locally for 24 hours; and (iii) control group - skin samples not treated with either ultrasound or the labeled complex. For each skin sample, three randomly selected rectangular cross-sections of the layers were used, and their pixel fluorescence intensity was calculated. The results are shown in Figure 7.

[0157] As shown in Figure 7, skin pretreated with ultrasound showed higher fluorescence intensity in the epidermis and dermis compared to skin not pretreated with ultrasound.

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

[0159] These results suggest that the combination of ultrasound pre-application and a carrier for HA delivery resulted in highly effective penetration of HA into deeper layers of the skin, including the target layer (dermis).

[0160] (Example 4) (Stability of the Q-starch / HA complex in the skin layer) To evaluate the stability of the Q-starch / HA complex in the skin layer under the treatment conditions described in Example 3 above, the carrier Q-starch was labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (green), and the HA was labeled with Hylite® Fluor647 (red), resulting in a complex Q-starch with a final concentration of 0.25 N / O and 260 mM of complexed HA. 5-DTAF -HA Hylite Fluor647 The cells were formed as described in Materials and Methods. The fixed specimens were stained with DAPI to evaluate intact nucleated cells in the skin layer beneath the SC (the SC contained dead anucleated cells). The stained specimens were sectioned and visualized using a confocal microscope.

[0161] Confocal images of porcine skin samples were acquired 24 hours after local administration of the labeled complex, either after a 5-minute ultrasound examination or without a preceding ultrasound examination. Exemplary confocal images are shown in Figures 8A to 8D.

[0162] As can be seen in Figures 8A-8B, without US pretreatment, Q-starch after 24 hours 5-DTAF -HA Hylite Fluor647 Local administration of the compound resulted in both green and red staining present in the SC in a similar pattern. Penetration of the labeled complex into the epidermis in skin samples pretreated with US application was observed in Q-starch. 5-DTAF Green staining and HA due to the presence of Hylite Fluor647 This is shown as red staining due to the presence of HA, and the green and red staining patterns are similar (Figures 8C-8D). These results indicate that HA does not deassemble from Q-starch, and the complex maintains its stability in the deeper skin layers.

[0163] (Example 5) (In vivo permeability test) To evaluate the uptake of the Q-starch-hyaluronic acid complex into the skin in mice in vivo, design the following study.

[0164] The mice were divided into the following groups: Group I: control – mice that received no treatment; Group II: mice treated with Q-starch-HA without US pretreatment; and Group III: mice administered with the Q-starch-HA complex (in different N / O molar ratios) after US pretreatment. Hyaluronic acid was administered to Hylite® Fluor647 amine dye (HA). Hylite Fluor647 Labeled with ), and brought into contact with Q-starch to form Q-starch-HA Hylite Fluor647 Form a complex. Apply the following steps in the research protocol: 1. Shave the back of each mouse using a hair clipper. Then, anesthetize the mice 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 chamber.

[0166] 3. Make an incision of approximately 1 cm near the tail. To measure the initial conductivity of the skin, one conductive electrode is placed inside the chamber and the other conductive electrode is placed inside the incision.

[0167] 4. Subsequently, replace the PBS in the chamber with 1% SLS in PBS, and measure the skin conductivity again.

[0168] 5. Group III mice were subjected to US treatment (QSonica Q700Sonicator, frequency = 20kHz, 6.1~10.5W / cm²). 2 (Probe diameter 1.3 cm), pretreatment was performed: The ultrasound probe was placed in a plastic cylinder 8 mm from the surface of the skin. 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 evaluate skin permeability, conductivity measurements were performed during ultrasound exposure. Ultrasound application was performed when conductivity was 50-70 times the initial conductivity, or 0.70 (kΩ*cm). 2 ) -1It turns off when it reaches a predetermined value.

[0169] 6. Remove the plastic cylinder from mouse groups II and III, and administer Q-starch-HA. Hylite Fluor647 Prepare the composite 40 minutes before US application and place it inside the rubber ring. Apply the Parafilm cover to prevent fluid leakage from the ring.

[0170] 7. Twenty hours after administration of the complex, mice from groups I-III were sacrificed, their skins were removed, fixed in 4% formalin, and cut into 5 μm slices (slides).

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

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

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

[0174] [Figure 1A]Figures 1A and 1B are graphs showing the size distribution of the quaternary starch (Q-starch) and hyaluronic acid (HA) complex (Q-starch-HA complex) (1A), as well as free Q-starch and HA (1B), obtained using dynamic light scattering (DLS). The Q-starch-HA complex is characterized by an increased ratio (N / O ratio) between the positively charged amine groups (N) of Q-starch and the negatively charged carboxyl groups (O) of the HA backbone. [Figure 1B] Figures 1A and 1B are graphs showing the size distribution of the quaternary starch (Q-starch) and hyaluronic acid (HA) complex (Q-starch-HA complex) (1A), as well as free Q-starch and HA (1B), obtained using dynamic light scattering (DLS). The Q-starch-HA complex is characterized by an increased ratio (N / O ratio) between the positively charged amine groups (N) of Q-starch and the negatively charged carboxyl groups (O) of the HA backbone. [Figure 2] Figure 2 is a graph showing the size distribution (average diameter) of free Q-starch, free HA, and Q-starch-HA complexes with an N / O ratio of 0.25, as measured using the NanoSight system. [Figure 3] Figure 3 is a bar graph showing the average ζ potential (effect of particle surface charge) of free HA, Q-starch, and Q-starch-HA composites, characterized by an increasing N / O ratio. [Figure 4A] Figures 4A–4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and newly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C–4G). [Figure 4B] Figures 4A–4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and newly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C–4G). [Figure 4C]Figures 4A–4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and newly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C–4G). [Figure 4D] Figures 4A–4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and newly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C–4G). [Figure 4E] Figures 4A–4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and newly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C–4G). [Figure 4F] Figures 4A–4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and newly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C–4G). [Figure 4G] Figures 4A–4G are exemplary Cryo-TEM images of free (uncomplexed) Q-starch (4A), free (uncomplexed) HA (4B), and newly prepared Q-starch-HA complexes at N / O molar ratios ranging from 0.25 to 3 (4C–4G). [Figure 5A]Figures 5A and 5B are bright-field confocal images of exemplary pig ear skin cross-sections after 24-hour incubation with 0.3% (w / v) HA labeled with Hylite® Fluor647 dye (HAHylite Fluor 647), either without ultrasound (US) pretreatment (5A) or after 5 minutes of US application (5B). The stratum corneum (SC), epidermis, and dermis are shown (bars: 20 μm). The fluorescence intensity of HAHylite Fluor 647, calculated for pixels in the exemplary rectangular cross-section shown in image j, is shown for each cross-section as a function of distance from SC to a depth of 200 μm. Vertical dashed lines represent interlayer separation. Horizontal dashed lines indicate skin autofluorescence at the wavelength of labeled HA. [Figure 5B] Figures 5A and 5B are bright-field confocal images of exemplary pig ear skin cross-sections after 24-hour incubation with 0.3% (w / v) HA labeled with Hylite® Fluor647 dye (HAHylite Fluor 647), either without ultrasound (US) pretreatment (5A) or after 5 minutes of US application (5B). The stratum corneum (SC), epidermis, and dermis are shown (bars: 20 μm). The fluorescence intensity of HAHylite Fluor 647, calculated for pixels in the exemplary rectangular cross-section shown in image j, is shown for each cross-section as a function of distance from SC to a depth of 200 μm. Vertical dashed lines represent interlayer separation. Horizontal dashed lines indicate skin autofluorescence at the wavelength of labeled HA. [Figure 6A]Figures 6A–6D are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with a labeled Q-starch / HA complex (Q-starch-HAHylite Fluor 647) characterized by an N / O molar ratio of 0.25. Skin samples were either not pre-treated with ultrasound before topical application of the labeled Q-starch / HA complex (6A, 6B) or treated with ultrasound for 5 minutes before application of the complex (6C, 6D). Figures 6A and 6C are confocal images showing intact nucleated cells in the dermal layer beneath 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). The fluorescence intensity of Q-starch-HAHylite Fluor 647 is calculated for pixels in the exemplary rectangular cross-section shown in image j as the 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. Vertical dashed lines represent interlayer separation. Horizontal dashed lines show the autofluorescence of the skin at the wavelength of labeled HA. [Figure 6B]Figures 6A–6D are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with a labeled Q-starch / HA complex (Q-starch-HAHylite Fluor 647) characterized by an N / O molar ratio of 0.25. Skin samples were either not pre-treated with ultrasound before topical application of the labeled Q-starch / HA complex (6A, 6B) or treated with ultrasound for 5 minutes before application of the complex (6C, 6D). Figures 6A and 6C are confocal images showing intact nucleated cells in the dermal layer beneath 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). The fluorescence intensity of Q-starch-HAHylite Fluor 647 is calculated for pixels in the exemplary rectangular cross-section shown in image j as the 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. Vertical dashed lines represent interlayer separation. Horizontal dashed lines show the autofluorescence of the skin at the wavelength of labeled HA. [Figure 6C]Figures 6A–6D are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with a labeled Q-starch / HA complex (Q-starch-HAHylite Fluor 647) characterized by an N / O molar ratio of 0.25. Skin samples were either not pre-treated with ultrasound before topical application of the labeled Q-starch / HA complex (6A, 6B) or treated with ultrasound for 5 minutes before application of the complex (6C, 6D). Figures 6A and 6C are confocal images showing intact nucleated cells in the dermal layer beneath 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). The fluorescence intensity of Q-starch-HAHylite Fluor 647 is calculated for pixels in the exemplary rectangular cross-section shown in image j as the 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. Vertical dashed lines represent interlayer separation. Horizontal dashed lines show the autofluorescence of the skin at the wavelength of labeled HA. [Figure 6D]Figures 6A–6D are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with a labeled Q-starch / HA complex (Q-starch-HAHylite Fluor 647) characterized by an N / O molar ratio of 0.25. Skin samples were either not pre-treated with ultrasound before topical application of the labeled Q-starch / HA complex (6A, 6B) or treated with ultrasound for 5 minutes before application of the complex (6C, 6D). Figures 6A and 6C are confocal images showing intact nucleated cells in the dermal layer beneath 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). The fluorescence intensity of Q-starch-HAHylite Fluor 647 is calculated for pixels in the exemplary rectangular cross-section shown in image j as the 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. Vertical dashed lines represent interlayer separation. Horizontal dashed lines show the autofluorescence of the skin at the wavelength of labeled HA. [Figure 7] Figure 7 is a bar graph showing the fluorescence intensity measured at the wavelength of 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 treated topically with labeled Q-starch-HA complex (Q-starch-HAHylite Fluor 647) for 24 hours; (ii) skin samples treated with ultrasound for 5 minutes, followed by topical application of Q-starch-HAHylite Fluor 647 for 24 hours; and (iii) a control group – skin samples not treated with ultrasound or the labeled complex. This group is useful for autofluorescence measurements. Fluorescence intensity was calculated by image j based on data recorded from confocal scanning (3 repeats ± SEM). [Figure 8A]Figures 8A–8D are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with labeled Q-starch / HA having an N / O molar ratio of 0.25, where Q-starch is labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch 5-DTAF) and appears as a light green stain in images 8A and 8C, and HA is labeled with Hylite® Fluor 647 (HAHylite Fluor 647) and appears as a red stain in images 8B and 8D. Intact cell nuclei under SC are stained blue (DAPI staining). Skin samples were either not pre-treated with ultrasound before topical administration of the labeled complex Q-starch 5-DTAF-HAHylite Fluor 647 (8A, 8B) or were treated with ultrasound for 5 minutes before complex application (8C, 8D). Bar: 20 μm. [Figure 8B] Figures 8A–8D are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with labeled Q-starch / HA having an N / O molar ratio of 0.25, where Q-starch is labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch 5-DTAF) and appears as a light green stain in images 8A and 8C, and HA is labeled with Hylite® Fluor 647 (HAHylite Fluor 647) and appears as a red stain in images 8B and 8D. Intact cell nuclei under SC are stained blue (DAPI staining). Skin samples were either not pre-treated with ultrasound before topical administration of the labeled complex Q-starch 5-DTAF-HAHylite Fluor 647 (8A, 8B) or were treated with ultrasound for 5 minutes before complex application (8C, 8D). Bar: 20 μm. [Figure 8C]Figures 8A–8D are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with labeled Q-starch / HA having an N / O molar ratio of 0.25, where Q-starch is labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch 5-DTAF) and appears as a light green stain in images 8A and 8C, and HA is labeled with Hylite® Fluor 647 (HAHylite Fluor 647) and appears as a red stain in images 8B and 8D. Intact cell nuclei under SC are stained blue (DAPI staining). Skin samples were either not pre-treated with ultrasound before topical administration of the labeled complex Q-starch 5-DTAF-HAHylite Fluor 647 (8A, 8B) or were treated with ultrasound for 5 minutes before complex application (8C, 8D). Bar: 20 μm. [Figure 8D] Figures 8A–8D are confocal images of exemplary pig ear skin sections histologically stained after 24-hour incubation with labeled Q-starch / HA having an N / O molar ratio of 0.25, where Q-starch is labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) (Q-starch 5-DTAF) and appears as a light green stain in images 8A and 8C, and HA is labeled with Hylite® Fluor 647 (HAHylite Fluor 647) and appears as a red stain in images 8B and 8D. Intact cell nuclei under SC are stained blue (DAPI staining). Skin samples were either not pre-treated with ultrasound before topical administration of the labeled complex Q-starch 5-DTAF-HAHylite Fluor 647 (8A, 8B) or were treated with ultrasound for 5 minutes before complex application (8C, 8D). Bar: 20 μm.

Claims

1. A complex of hyaluronic acid and chemically modified polysaccharides.

2. The complex according to claim 1, wherein the polysaccharide is modified by substitution of one or more positively charged chemical moieties.

3. The composite according to claim 2, wherein the positively charged chemical moiety is a quaternary amine group.

4. The complex according to claim 1, wherein the polysaccharide is selected from the group consisting of starch, chitosan, pectin, cellulose, dextran, and galactan.

5. The composite according to claim 4, wherein the polysaccharide is starch.

6. The hyaluronic acid mentioned above is (CH ３ ) ３ -N ＋ The complex according to any one of claims 1 to 5, wherein it is complexed with starch substituted with -.

7. The composite according to claim 2, wherein the molar ratio (N / O molar ratio) of the positively charged chemical portion of the modified starch to the negatively charged carboxyl group of hyaluronic acid is in the range of about 0.20 to about 3.00, or about 0.25 to about 1.

5.

8. A composition comprising a complex of hyaluronic acid and a chemically modified polysaccharide according to any one of claims 1 to 7, and a physiologically acceptable excipient.

9. The composition according to claim 8, which is incorporated as a cosmetic composition.

10. The composition according to claim 8, which is formulated as a therapeutic composition.

11. (a) at least one complex of hyaluronic acid and a chemically modified polysaccharide as described in any one of claims 1 to 7, or a composition comprising such complex; (b) means for applying ultrasonic treatment; and, (c) Optionally, instructions and means for administering the complexed hyaluronic acid and / or the composition to a subject. A kit that includes this.

12. A complex, composition, or kit according to any one of claims 1 to 11 for use in enhancing the non-invasive transdermal delivery of hyaluronic acid.

13. The complex, composition, or kit according to claim 12, wherein the hyaluronic acid is high molecular weight hyaluronic acid.

14. A pharmaceutical composition comprising a glycosaminoglycan (GAG) as an active ingredient, used for non-invasive transdermal delivery of a GAG to a target, wherein the transdermal delivery includes the application of ultrasound to the skin surface of the target 5 seconds to 5 minutes before the use of the pharmaceutical composition.

15. The pharmaceutical composition according to claim 14, wherein the GAG ​​is complexed with at least one chemically modified polysaccharide as described in claims 1 to 6.

16. The pharmaceutical composition according to claim 14, wherein the use of the pharmaceutical composition is repeated at least once after the application of ultrasound.

17. The pharmaceutical composition according to any one of claims 14 to 16, wherein the GAG ​​is selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate.

18. The pharmaceutical composition according to claim 14, wherein ultrasound is applied for 30 seconds to 9 minutes.

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