Surface modified dendrimer for controlled release of active ingredients into the skin

Abstract

A modified nanoparticle nanocarrier system for delivering one or more active ingredients into a skin that can slowly release the active ingredients is provided. The modified nanoparticle nanocarrier system comprises a dendrimer, a linker and a peptide, wherein the dendrimer is first coated with the linker to form a dendrimer-linker conjugate, and the dendrimer-linker conjugate is then coupled to a peptide to form a dendrimer-linker-peptide conjugate. Subsequently, the dendrimer-linker-peptide conjugate is loaded with one or more active ingredients and mixed with a topical base to form a homogenized product to be applied onto the skin.

Description

[0001] SURFACE MODIFIED DENDRIMER FOR CONTROLLED RELEASE OF ACTIVE INGREDIENTS INTO THE SKIN

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to and the benefits of U.S. Provisional Application No. 63 / 686,886 filed on August 26, 2024, the content of which is incorporated in its entirety by reference herein.

[0004] BACKGROUND OF THE INVENTION

[0005] The skin is the largest organ in the body and it serves as the first line of defense against harmful external substances. It protects the body from external factors such as chemicals, temperatures, or bacteria. The skin undergoes a variety of changes due to exposure to these external factors. As the skin ages, the skin becomes thinner, more fragile, and loses elasticity. The production of oil and blood flow in the skin decreases, while the number of skin cells also reduces. [1] The skin also changes due to internal factors. The skin's ability to repair damage slows down, leading to a decrease in collagen and elastin production. Thus, the skin needs constant supplementation of active ingredients to keep the skin healthy and firm, and particularly external supplementation with biologically active ingredients to improve skin health. [2] Active ingredients may be biological active ingredients which selectively treat skin problems. However, active ingredients may cause skin irritation if burst released into the body.

[0006] In general, skincare is divided into cosmetics, which conceal issues over the skin with dyes and pigments and so-called “cosmeceuticals,” which deliver pharmacologically active ingredients into the skin. [3] Active ingredients may include biologically active substances used in cosmeceutical formulas to achieve local cellular effects. Active ingredients improve skin health by reducing signs of aging and treating skin dryness, pigmentation issues, and acne. However, some of the active ingredients in skincare products can cause local skin irritation and peeling of the stratum corneum if allowed to accumulate in this part of the skin. [4] Embodiments of the present invention include a novel nanocarrier, Dendrimer-Linker- Peptide (DE-Linker-PE), e.g., Dendrimer-Polyoxazoline-Peptide (DE-POZ-PE), which can slowly release active ingredients at specific conditions such as temperature and pH. When an active ingredient is encapsulated in the nanocarrier it may release for an extended time (24h) at body temperature (37°C).

[0007] Additionally, it is crucial for active ingredients to penetrate the dermis for effective localization within the extracellular matrix. In vitro studies show that the nanocarrier according to the preferred embodiment in the current invention can deliver cargo to the dermis layer of the skin. Moreover, it is also shown that the nanoparticles are not toxic and biocompatible with skin fibroblast cells.

[0008] FIELD OF THE INVENTION

[0009] Embodiments of the present invention relate to structures and synthesis of a novel nanocarrier, Dendrimer-Linker-Peptide (DE-Linker-PE), such as Dendrimer-Polyoxazoline- Peptide (DE-POZ-PE). Further embodiments of the invention relate to loading the nanocarrier with one or more active ingredients, a nanocarrier-active ingredient complex, and / or transdermal delivery of the active ingredient. In some embodiments, the delivery of the active ingredient is triggered by temperature and / or pH. In some embodiments, the delivery may be prolonged. By way of example, some embodiments of the present invention relate to external delivery of macromolecules to the skin cells through tropical application of nanocarrier-active ingredient formulations and related delivery products.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Various aspects of the present invention are illustrated by way of example, and not by way of limitation, in the accompanying drawings, wherein:

[0012] Fig. 1 depicts an illustration showing modified dendrimer nanoparticle penetrating different layers of skin in accordance with the principles of the present invention;

[0013] Fig. 2A depicts tosylate activation of polyoxazoline (POZ) step of a synthesis route of DE-POZ-PE in accordance with the principles of the present invention; Fig. 2B depicts conjugation of POZ-OTs on the surface of G4-PAMAM dendrimer (DE) step of the synthesis route of DE-POZ-PE in accordance with the principles of the present invention;

[0014] Fig. 2C depicts clicking of azide-modified peptide (PE) to the surface of Dendrimer- Poly oxazoline (DE-POZ) to obtain final product in the synthesis route of DE-POZ-PE in accordance with the principles of the present invention;

[0015] Fig. 2D depicts the Dendrimer-Polyoxazoline-Peptide (DE-POZ-PE) in accordance with the principles of the present invention;

[0016] Fig. 2E depicts the preparation of Poly(2-ethyl-2-oxazoline), alkyne terminated with p- Toluenesulfonyl chloride;

[0017] Fig. 2F depicts a further detailed view of the formation of DE-POZ reaction of Fig. 2B;

[0018] Fig. 2G depicts synthesis of palmitoyl pentapeptide-4-Boc;

[0019] Fig. 2H depicts synthesis of 5-azido pentanol;

[0020] Fig. 21 synthesis of palmitoyl pentapeptide-4-Boc-ester-azide;

[0021] Fig. 2J depicts synthesis of DE-POZ-PE;

[0022] Fig. 2K depicts synthesis of 3 -azido-propanol;

[0023] Fig. 2L depicts synthesis of Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA);

[0024] Fig. 3 A depicts Matrix-Assisted Laser Desorption / Ionization Time of Flight (MALDI- TOF) of DE-POZ in accordance with the principles of the present invention;

[0025] Fig. 3B depicts MALDI-TOF of DE-POZ-PE in accordance with the principles of the present invention;

[0026] Fig. 3C depicts Dynamic Light Scattering Spectroscopy (DLS) of DE, DE-POZ, and DE- POZ-PE in accordance with the principles of the present invention;

[0027] Figure 3D depicts H NMR of Poly(2-ethyl-2-oxazoline)-tosylchloride;

[0028] Figure 3E depicts NMR of palmitoyl pentapeptide-4-Boc;

[0029] Figure 3F depicts NMR of 5-azido pentanol;

[0030] Figure 3G depicts 'H NMR of palmitoyl pentapeptide-4-Boc-ester-azide; Figure 3H depicts ES MS of palmitoyl pentapeptide-4-Boc-ester-azide;

[0031] Figure 31 depicts 'H NMR of 3 -azido-propanol;

[0032] Figure 3 J depicts 'H NMR of Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA);

[0033] Fig. 4A depicts a visual representation of conduction of the release study experiments in accordance with the principles of the present invention;

[0034] Fig. 4B depicts a release of retinol from DE in accordance with the principles of the present invention;

[0035] Fig. 4C depicts a release of retinol from DE-POZ in accordance with the principles of the present invention;

[0036] Fig. 4D depicts a release of retinol from DE-POZ-PE in accordance with the principles of the present invention;

[0037] Fig. 5 depicts the loading efficiency of all-trans retinol in three different systems: DE, DE-POZ, and DE-POZ-PE;

[0038] Fig. 6A depicts a graph of the cell viability of DE in accordance with the principles of the present invention;

[0039] Fig. 6B depicts a graph of the cell viability of DE-POZ in accordance with the principles of the present invention;

[0040] Fig. 6C depicts a graph of the cell viability of DE-POZ-PE in accordance with the principles of the present invention;

[0041] Fig. 7A depicts a skin penetration study on naive porcine skin imitating human skin texture in accordance with the principles of the present invention;

[0042] Fig. 7B depicts a skin penetration study on porcine skin incubated Cy5 in accordance with the principles of the present invention;

[0043] Fig. 7C depicts a skin penetration study on porcine skin incubated with DE(Cy5) in accordance with the principles of the present invention;

[0044] Fig. 7D depicts a skin penetration study on porcine skin incubated with DE-POZ(Cy5) in accordance with the principles of the present invention; Fig. 7E depicts a skin penetration study on porcine skin incubated with DE-POZ- PE(Cy5) in accordance with the principles of the present invention;

[0045] Fig. 8A-8E depict Figs. 7A-7E in color; and

[0046] Fig. 9 depicts a graph of human dermal fibroblasts incubated with different treatment, such as, cells only without any stimulants, stimulants such as TGF- 1 and ascorbic acid which stimulate collagen production. Next all-trans retinol (RE), DEPOZ(RE), PE and DE-POZ-PE. After incubation cells were stained with picro-sirius red dye to visualize collagen production intensity in each sample in accordance with the principles of the present invention.

[0047] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The detailed description set forth below in connection with the appended drawings is intended as a description of various embodiments of the invention and is not intended to represent the only embodiments in which the invention may be practiced. The detailed description includes specific details for the purpose of providing a thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without these specific details. In some instances, well known structures and components are shown in block diagram form in order to avoid obscuring the concepts of the invention.

[0049] Aspects of the present invention are described herein with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems) and structures according to embodiments of the invention. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented as one or more steps of a method. In some steps, the method may be performed by a computer, such as, when measuring Fourier-transform spectroscopy (FT-IR), Nuclear Magnetic Resonance spectroscopy (NMR), powder X-ray diffraction spectroscopy (PXRD), etc. These computer program instructions may be provided to a processor of a controller, a general purpose computer, a special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which executed via the processor of the controller or other programmable data processing apparatus, create means for implementing the functions / acts specified in the flowchart and / or block diagram block or blocks. The output may be displayed in a graph on a scale that would be interpretable by one having ordinary skill in the art.

[0050] The following description is provided to enable any person skilled in the art to practice the various embodiments described herein. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments. Thus, the claims are not intended to be limited to the embodiments shown herein, but are to be accorded the full scope consistent with each claim’s language, wherein reference to an element in the singular is not intended to mean “one and only one” unless specifically so stated, but rather “one or more.” Similarly, references to an element in the singular in the description mean “one or more” unless specifically stated otherwise. All structural and functional equivalents to the elements of the various embodiments described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. No claim element is to be construed under the provisions of 35 U.S.C. §112, sixth paragraph, unless the element is expressly recited using the phrase “means for” or, in the case of a method claim, the element is recited using the phrase “step for.”

[0051] The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The description of the present invention has been presented for purposes of illustration and description but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Aspects of the invention were chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.

[0052] With respect to the present application, “congener” means a chemical constituent of the same kind or category, having similar chemical properties, binding affinity, and functionality, and minimal structural changes, relative to the referenced chemical compound. With respect to the present application, “about” or “approximately” means within plus or minus one at the last reported digit. For example, about 1.00 means 1.00 ± 0.01 unit.

[0053] With respect to the present application, “around” used in conjunction with a numeral measurement means within plus or minus one unit. For example, around 50% means 49% - 51%. For example, around 11.01 units means 10.01 - 12.01.

[0054] With respect to the present description “and” and “or” shall be construed as conjunctively or disjunctively, whichever provides the broadest disclosure in each instance of the use of “and” or “or.”

[0055] Embodiments of the present invention relate to structures and synthesis of a novel nanocarrier Dendrimer-Linker-Peptide (DE-Linker-PE), such as Dendrimer-Polyoxazoline- Peptide (DE-POZ-PE). Further embodiments of the invention relate to loading the nanocarrier with one or more active ingredients, a nanocarrier-active ingredient complex, and / or transdermal delivery of the active ingredient. In some embodiments, the delivery of the active ingredient is triggered by temperature and / or pH. In some embodiments, the delivery may be prolonged. By way of example, some embodiments of the present invention relate to external delivery of macromolecules to the skin cells through topical application of nanocarrier-active ingredient formulations and related cosmetic and delivery products.

[0056] In the following description, active ingredients may include pharmaceuticals, nutrients, vitamins, short peptides, and plant extracts, neutraceuticals, and derivatives and congeners thereof, and / or mixtures thereof for skincare formulations. Example topical cosmetic active ingredients can be an alpha or beta hydroxy acid, anti-wrinkle agent, anti-aging agent, skinlightening agent, anti-dark circle agent, peptide, amino acid, plant extracts, vitamin, antioxidant, anti-inflammatory agent, humectant, keratolytic agent, antibacterial agent, antifungal agent, a sunscreen agent, of which may include but not limited to niacinamide and resveratrol, glycolic acid, lactic acid, salicylic acid, gluconolactone, lacftobionic acid, citric acid, hyaluronic acid, sodium hyaluronate, retinol, retinyl palmitate, panthenol, allantoin, ceramide, caffeine, ubiquinone, kojic acid, hydroquinone, ascorbic acid, ascorbyl glucoside, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, acetyl hexapeptide-8, acetyl hexapeptide-3, palmitoyl tripeptide-38, palmitoyl tripeptide-1, palmitoyl tripeptide-5, hydrolyzed rice protein, bakuchiol, camellia sinensis leaf extract, centella asiatica extract, citrus aurantium dulcis fruit extract, citrus limon fruit extract, ferulic acid, ginkgo biloba leaf extract, glyceryl linoleate, glyceryl linolenate, lyceum barbarum fruit extract, oat amino acids, tocopherol, tocopheryl acetate, vitis vinifera leaf extract, lipoic acid, folic acid, coffea arabica seed extract, and cucumis sativus fruit extract.

[0057] For these active ingredients to work, they must be delivered to the correct location. That is, the active ingredient must penetrate the epidermis to be released in the dermis. Furthermore, concentrated release of certain active ingredients at the stratum corneum may damage the epidermis. The skin is a multi-layered organ with each layer serving a function. The outermost layer of the skin, the epidermis, is composed of stratified squamous epithelium and is avascular. The epidermis includes several sub-layers (stratum corneum, lucidum, granulosum, spinosum, basale) that facilitate cellular renewal from the basal layer to the surface. This layer is a protective barrier against environmental hazards (pathogens, chemicals, UV radiation) and prevents dehydration. It also contains melanocytes for UV protection and pigmentation, Langerhans cells for immune response, and Merkel cells for sensory reception.

[0058] The stratum corneum, the outermost layer of the epidermis, is composed of flattened, dead keratin-rich cells (corneocytes) embedded in a lipid matrix, serving as a vital barrier to protect against environmental stressors and prevent water loss. This layer contains no living tissue, so it is not a viable target for active pharmaceutical ingredients. Further, this layer, being relatively impermeable to water and yet oleophilic, inhibits the movement of drugs deeper into the skin while allowing them to accumulate.

[0059] Beneath the epidermis lies the dermis, a dense layer of connective tissue rich in collagen and elastin fibers, providing the skin with strength and elasticity. It is highly vascularized and supports various skin appendages (hair follicles, sweat, and sebaceous glands) and nerve endings. The dermis plays a critical role in maintaining the skin's structural integrity, enabling sensations, thermoregulation through sweat glands, and nourishment to the skin. It also facilitates oil secretion for skin lubrication and barrier function.

[0060] Some of the active ingredients in skincare products can cause local skin irritation and peeling of the stratum corneum if allowed to accumulate in this part of the skin. [4] A possible solution to enable slow-release delivery of these active ingredients into the skin is via a nanocarrier system, which can enhance follicular openings, resulting in better skin penetration.

[0061] An effective drug delivery system should not only penetrate the stratum comeum but also release the drug slowly in the deeper layers of the skin. The properties should not only minimize skin irritation but also ensure a sustained therapeutic effect throughout the day. Hence, improving dermal delivery and penetration is essential to provide an even distribution of active ingredients. [5]

[0062] Nanocarriers are useful transport agents that can be fine-tuned to modify their physical and biological properties to carry therapeutics or active ingredients to targeted tissue. [6] The stratum corneum, which has a thickness of 10 pm, is the primary route through which nanoparticles can penetrate the skin. [7] Nanoparticles should be less than 100 nm and as lipophilic as possible so they can fit into the inter-corneocyte spaces of the skin. [8] Nanoparticles must balance their efficacy in delivering materials to their location with controlled release and cytotoxicity.

[0063] There are several ways to deliver active ingredients into the skin, such as chemical permeation enhancers that include solvents, terpenes, fatty acids, surfactants, and other chemical agents that interfere with the skin lipids and enhance skin penetration of active ingredients. [9] In addition, scientists have tried to develop more well-defined delivery particles such as microsponges, silica nanoparticles, and lipid nanovesicles. While effective in topical applications, microsponges are limited by their micron-sized range. [10, 11]

[0064] Silica nanoparticles have demonstrated improvements in cosmetic product effectiveness, texture, and shelf life; however, their use is hindered by concerns over toxicity.

[0012]

[0065] Lipid Nanovesicles are vesicular systems composed of one or more phospholipid bilayers, separated by an aqueous compartment; however, drawbacks include low drug loading efficiency, the potential for drug leakage, and low stability at room temperature.

[0013]

[0066] Embodiments of the present invention overcome the challenges of known nanocarriers through a DE-Linker-PE conjugate complexed with one or more active ingredients. Fig. 1 depicts an illustration showing modified dendrimer nanoparticle nanocarrier 10 penetrating different layers of skin in accordance with the principles of the present invention. As illustrated, the different layers of skin consist of the epidermis layer 8, the dermis layer 6, and the subcutaneous layer 4, all external to the muscle 2. The epidermis layer 8 further consists of the sublayers stratum basale 8a, stratum spinosum 8b, stratum granulosum 8c, stratum lucidum 8d, and stratum corneum 8e. To overcome the challenges of known nanocarriers, embodiments of the present invention include a nanocarrier 10 that may slowly release the active ingredients at specific conditions — pH and temperature — and may simultaneously deliver active ingredients into the dermis layer 6 of the skin.

[0067] Embodiments of the DE-Linker-PE nanocarrier 18 of the present invention include a dendrimer (DE) 12, a linker 14, and a peptide (PE) 16. Dendrimers 12 are a class of polymers which are also known as starburst polymers due to the shapes of these polymers. In general, a dendrimer 12 exhibits a tree-like molecular architecture with an interior core 12a, interior layers (also called "generations”) 12b which consist of repeating units regularly attached to the core 12a, and an exterior surface of functional terminal groups 12c attached to the outermost generation. Generation number is determined by the number of focal points going from the core 12a towards the surface.

[0068] Dendrimers 12 have a uniform size and shape, which is critical for consistent performance in applications like drug delivery. The branched architecture provides a high degree of surface functionality, allowing for the attachment of multiple therapeutic or diagnostic agents. The precise molecular weight and size control make dendrimers predictable in their behavior and interaction with biological systems. Many dendrimers 12 are soluble in water and other solvents, enhancing their usability in various formulations. The type and density of functional groups 12c on the surface can be tailored to enhance targeting, binding, and solubility. The core 12a of the dendrimer 12 can influence its overall stability and solubility. Higher-generation dendrimers 12 have more surface groups and larger size, which can improve active ingredients loading capacity but may also affect solubility and toxicity.

[0069] According to the preferred embodiments, the dendrimeric structure can comprise linear polymer structures as well as branched polymer structures. According to some preferred embodiments, the linear polymer, which is attached to said dendrimer 12, may be selected from the group consisting of polyamino acid, such as polyglycine, polylysine, polytyrosine and polyphenylalanine, dextran, polysaccharides, polypropylene oxide (PPO), poly D-amino acids, a copolymer of polyethylene glycol (PEG) with PPO, PEG, polyglycolic acid, polyvinyl pyrolidone, polylactic acid and polyvinylalcohol, or a mixture thereof. The length of said linear polymer may preferably be about 3 to about 20 atoms, e.g., the backbone atoms in the linear polymer, such as 3, 4, 5, 6, 8, 10, 15, or 20 atoms. Preferably, the group linking the linear polymers to said dendrimeric structure may be selected from the group consisting of amides, carboxylic acid esters, thioesters, disulfides, thiourethanes, carbamates, carbonates, thioureas or ureas.

[0070] According to the preferred embodiments, non-limiting examples of such suitable dendrimers 12 of the present invention include PAMAM (poly (amidoamine)), PAMAMOS (poly (amidoamine - organosilicon)), DAB (diaminobutanepoly (propylene imino), PPI (poly (propylene imine)), amphiphilic dendrimer, chiral dendrimer, multilingual dendrimer, micellar dendrimer, Tecto dendrimer and Frechet type dendrimer. The dendrimer 12 may be GO to GIO.

[0071] Embodiments of the present invention may comprise a biocompatible polymer and peptide-coated G4-PAMAM dendrimers 22, with each component designed to control drug release, biocompatibility, and tissue localization. As previously stated, dendrimers 12 are well- defined versatile macromolecules composed of a central core 12a with branches / generations 12b radiating outward and terminal functional groups 12c on the periphery.

[0014]

[0072] As monomers are added to branching units 12b connected to the core 12a of the dendrimer 12, the dendrimer 12 grows. For example, according to one embodiment, the polyamidoamine (PAMAM) dendrimer 20 size increases by 1 nm for each monomer length added to each of the of the added branches. By way of example, GO has 4 branches, G1 has 8, G2 has 16, G3 has 32, and G4 has 64 branches. Dendrimers 12 add around 1 nm size increase by increased generation 12b. Overall, size can vary from 1 nm to 12 nm as they grow in generation (number of branch units) 12b from one to ten.

[0015] Internal amide bonds and exterior amine groups make the structure of PAMAM dendrimers 20 resemble proteins. Based on their systematic structure and electrophoretic properties, they are often called “artificial proteins.”

[0016] The interior hydrophobic cavities 12d of dendrimers 12 allow encapsulation of hydrophobic active ingredient or drugs, thereby increasing the solubility of these drugs in water.

[0017] In addition, this host-guest encapsulation of the drugs gives them an unimicellar nature.

[0018] However, the loading method is the same for hydrophilic active ingredient, although there may be a difference in loading efficacy. In embodiments where the cavity is hydrophilic 12e, the encapsulation rate of hydrophilic active ingredients may be higher than that of hydrophobic.

[0073] A study reported that G4 (fourth generation) PAMAM dendrimers 21 with -NH2 terminated groups could penetrate the skin better than -COOH and -OH periphery groups.

[0019] However, early studies on PAMAM dendrimers found terminal NH2 groups could induce cytotoxicity in some cell lines, as the terminal amines can disrupt cellular membranes, causing cell lysis.

[0020]

[0074] This issue, unfortunately, has created a false impression that dendrimers 12 are not viable systems in drug delivery. Presently, two dendrimer-based drug delivery systems have since been approved by the FDA, and multiple clinical trials that use dendrimers are underway.

[0021] Many of these systems target diseases of the skin and mucosa, highlighting the translatability of this work beyond skin care and into areas related to treating cancer and infection. Furthermore, embodiments of the present invention show the nanocarrier to be an effective and safe drug delivery system using dendrimers 12.

[0075] The essential breakthrough that helped overcome issues with the toxicity of dendrimers was the realization that the toxic effects may be passivated by buffering cells from the positive charge of these amines using polyethylene glycol (PEG). However, consumer sentiment and well-founded concerns over PEG-related sensitization have driven the industry toward biocompatible alternatives. [22, 23]

[0076] Embodiments of the present invention include a linker 14 to overcome these challenges. Linkers 14 may be bound to the dendrimer 12 for modification of dendrimer 12 properties and for further attachment of one or more peptides 16 to form the nanocarrier, DE-Linker-PE 18. Thus, the linker 14 may be the connector between the dendrimer 12 and the peptide(s) 16. According to preferred embodiments, examples of suitable linkers 14 may include Polyoxazolines (POZ), Polyethylene Glycol (PEG), Poly(N-vinylpyrrolidone) (PVP), Poly(N- isopropyl acrylamide) (PNIPAM), Polysaccharides, Poly(lactic-co-glycolic acid) (PLGA), etc.

[0077] According to some preferred embodiments, linkers 14 may be cleavable linkers 14a (e.g., at pH change, enzymatic action), noncleavable linkers 14b, or biodegradable linkers 14c. Further, linkers 14 can preferably be selected based on hydrophilicity or hydrophobicity. Linkers 14 may be of any usable length. In addition, linkers 14 may preferably be selected to be nontoxic and biocompatible to avoid unwanted immune response and / or side effects.

[0078] Embodiments of the present invention may preferably include Polyoxazolines (POZ) 24 as the linker 14, which are nonionic, highly soluble in water and organic solvents, and stable polymers. Since the 1980s, this polymer has had a longstanding presence in the market and has been employed as a food additive, underscoring its safety. It has found applications in pharmaceutical and medicinal contexts, including conjugation to proteins, grafting in liposomes, and formulation into micelles. [24, 25] Polyoxazolines 24 are easily modified chemically by attaching leaving groups to conjugate them to proteins, enzymes, and different biological materials.

[0026]

[0079] While polyoxazolines 24 help overcome issues with cytotoxicity, skin penetration of these systems can be modest. So, to further enhance skin penetration, in a preferred embodiment, the system was modified with palmitoyl pentapeptide 26, which has been shown to penetrate all the skin layers and also has great stability in the skin against serine proteases.

[0027]

[0080] In addition to promoting skin penetration, this peptide 26 itself has been shown to stimulate ECM production, fibronectin, and type I and III collagen, and elastin and also stabilize mRNA, which enables TGF-synthesis.

[0028]

[0081] Furthermore, it significantly increases procollagen production and regulates hyaluronic acid synthesis by human fibroblasts.

[0029]

[0082] Preferably, embodiments of the present invention include a nanocarrier system 10 that allows for the slow-release delivery of all -trans retinol 28 within the dermis 6 of model porcine skin. According to the preferred embodiments, the nanocarrier system! 8 is DE-POZ-PE 30, which may comprise a G4-PAMAM dendrimer 21, poly(2-ethyl-2-oxazoline) 24, and palmitoyl pentapeptide-4 26 in some embodiments.

[0083] It is demonstrated that the system is pH and temperature-sensitive. At pH 5.0 and 37 °C, the release of all-trans-retinol 28 in all modified systems occurred at a slow rate, reaching maximum release within 24 hours. However, at pH 7.4 and 25 °C, no release of all-trans retinol 28 was observed.

[0084] The entire conjugated system, DE-POZ-PE 30, and DE-POZ 32 poses virtually no cytotoxicity relative to the base dendrimer 21, which allows for the safe uptake of the nanocarrier system 10 into the skin. Moreover, it was shown that the penetration of the nanocarrier system 10 to the dermis layer 6 of the skin allows for the codelivery of retinol 28, allowing for the maximum efficacy of the active ingredient.

[0085] Figs. 2A - 2D depict an overview of the synthesis of the DE-LINKER-PE 18, such as DE-POZ-PE 30.

[0086] Fig. 2A depicts tosylate activation of polyoxazoline (POZ) 24 step of a synthesis route of DE-POZ-PE 30 in accordance with the preferred embodiment under the principles of the present invention.

[0087] In this illustration, commercially available alkyne terminated Poly(2-ethyl-2-oxazoline) 24 with an average molecular weight of 5000 g / mol and terminating in a hydroxyl group was converted to a tosyl leaving group so that POZ-OTs 25 can react with amine on the dendrimer chains. (Figure 2A).

[0088] A leaving group is an atom or group of atoms that detaches from a molecule during a chemical reaction, typically a substitution or elimination reaction. Tosylate (p-toluenesulfonate, TsO ) is a well-known leaving group due to its stability and the ability to stabilize the negative charge after departure. Other suitable leaving groups may include Mesylate (MsO ), Triflate (TfO ), Iodide (I"), Bromide (Br j Chloride (CF) Acetate (AcO ). The synthesis of the conjugated nanocarrier involves three steps — 1) PAMAM-NH2 G4 dendrimer 21 is firstly coated in a tosylated polyoxazoline polymer 25 to increase molecular weight and improve colloidal stability. This is further functionalized with enzymatically digestible peptides to give the final product. (Figure 2)

[0089] The tosyl displacement with the primary amines of dendrimer 21 was performed under basic conditions to quench the build-up of p-toluenesulfonic acid during the reaction. (Figure 2F).

[0090] 2) Next, palmitoyl pentapeptide 26 was modified via (3-Dimethylamino-propyl)-ethyl- carbodiimide / N,N-Diisopropylethylamine (EDC / DIPEA) esterification reaction with 5-Azido-l- pentanol to obtain an enzymatically cleavable peptide 27. (Figure 21) Alternatively, 3-azido- propanol may also be used.

[0091] 3) Finally modified palmitoyl pentapeptide 27 was coupled to the alkyne terminating group of polyoxazoline 32 via click chemistry reaction at room temperature. (Figure 2J).

[0092] Synthesis of DE-POZ

[0093] Fig. 2B depicts conjugation of POZ-OTs 25 on the surface of G4-PAMAM dendrimer (DE) 21 step of the synthesis route of DE-POZ-PE 30 in accordance with the principles of the present invention. Dendrimers' sizes range from 1 nm to 15 nm, which is an important feature for a delivery system to have. This size range allows them to penetrate the skin more efficiently. For instance, G1 and G2 (first and second-generation) dendrimers have an excellent penetration due to their smaller size (less than 3 nm), but their drug loading efficiency is lower than that of third- or fourth-generation dendrimers. Hence, generation 4 of polyamidoamine dendrimer (G4- PAMAM) 21 was chosen as an option among other dendrimers 12 because of its optimal size (5nm), providing advantages in drug encapsulation and delivery applications.

[0094] Synthesis of DE-POZ-PE

[0095] Fig. 2C depicts clicking of azide-modified peptide (PE) 27 to the surface of Dendrimer- Polyoxazoline (DE-POZ) 32 to obtain final product in the synthesis route of DE-POZ-PE 30 in accordance with the principles of the present invention. Embodiments of the present invention include palmitoyl pentapeptide 26 as the peptide. Palmitoyl pentapeptide 26 is a skin penetration enhancer that may penetrate to the dermis and release active ingredient(s) at the dermis. Palmitoyl pentapeptide 26 is also useful in skincare preparations for its anti-aging properties.

[0096] According to preferred embodiments, examples of suitable peptides 16 include Palmitoyl Pentapeptide, Acetyl Hexapeptide-8, Palmitoyl Tripeptide-1, Palmitoyl Tetrapeptide-7, Copper Tripeptide-1, Palmitoyl Oligopeptide, Hexapeptide-11, Dipeptide Diaminobutyroyl Benzylamide Diacetate, Palmitoyl Tripeptide-5, Tripeptide-1, Pentapeptide- 18, Palmitoyl Hexapeptide- 12, Tripeptide-10 Citrulline, Acetyl Tetrapeptide-2, Acetyl Octapeptide-3, Myristoyl Pentapeptide- 17, Palmitoyl Tripeptide-38, Acetyl Dipeptide-1 Cetyl Ester, Tripeptide-3, Pentapeptide-3, Palmitoyl Hexapeptide- 19, Tetrapeptide-21, Oligopeptide-24, Palmitoyl Dipeptide-5, Dipeptide- 2, Tetrapeptide-2, Acetyl Hexapeptide-30, Acetyl Tetrapeptide-5, Acetyl Tetrapeptide-9, Palmitoyl Tripeptide-28, Oligopeptide-20, Tripeptide-5, Nonapeptide- 1, Tripeptide-32, Acetyl Tetrapeptide- 11, Acetyl Tetrapeptide-3.

[0097] Further suitable peptides 16 may have the following properties. 1) The peptides 16 may contain peptide bonds, which are amide linkages between amino acids. The peptide backbone is the primary structure determining their stability and functionality.

[0098] 2) The peptides 16 may have both hydrophilic (water-attracting) and hydrophobic (waterrepelling) regions, making them amphiphilic. This property allows them to interact with both water-based and lipid-based environments in the skin.

[0099] 3) The peptides 16 may form specific secondary structures, like alpha-helices or betasheets, depending on their amino acid sequence. This structural conformation can affect their biological activity.

[0100] 4) Suitable molecular weight for the peptides 16 may range from a few hundred to a few thousand Daltons.

[0101] Fig. 2D depicts the Dendrimer-Linker-Peptide 18, Dendrimer-Polyoxazoline-Peptide (DE-POZ-PE) 30 in accordance with the principles of the present invention. Varying quantities of polyoxazoline 24 were successfully conjugated to the dendrimer 21 , achieving the maximum attachment of 32 polyoxazoline units on a single dendrimer 21 (Figure 3 A).

[0102] Subsequently, the modified peptide 27 was attached, successfully achieving the conjugation of up to 13 peptide units within the dendrimer-polyoxazoline system 32. (Figure 3B).

[0103] Surface modification of G4-PAMAM dendrimer 21 increased its size from 5 nm (DE) 21 to 7nm (DE-POZ) 32 to finally 10 nm (DE-POZ-PE) 30 (Figure 3C). This shows that the size is still smaller than 100 nm, which is suitable to pass through the skin pores.

[0104] Modification of Polyoxazoline (POZ) 24 and Peptide (PE) 16 were confirmed by NMR (Figure 3D and 3F) and conjugation of DE-POZ 32 and DE-POZ-PE 30 were characterized by MALDI-TOF (Figure 3A and 3B).

[0105] Example Experimental Procedures for Synthesis of DE-POZ-PE

[0106] Figures 2E - 2L depict further details of the synthesis of DE-POZ-PE 30.

[0107] Material and Methods

[0108] Poly(2-ethyl-2-oxazoline), alkyne terminated 24 was purchased by Sigma-Aldrich. G4- PAMAM dendrimer 21 was purchased from Dendritech Inc. Palmitoyl pentapeptide-4 26 was purchased from XI’AN HUAWAVE BIOTECH CO., LTD. Piero Sirus Red dye was purchased from Mercedes Scientific. Primary Dermal Fibroblast: Normal, Human, Adult (HDFa) cells were purchased from ATTC, cat. number PCS-201-012. Porcine skin was purchased from Sierra for Medical Science. LDH-Cytotoxicity Assay Kit was purchased from BioLegend. CRYOSEAL 60 was purchased from Electron Microscopy Sciences. Differentiation Solution (Accumate) was purchased from Sigma-Aldrich. A modified Harris hematoxylin solution was purchased from Sigma- Aldrich. DAPI solution was purchased from Sigma-Aldrich. BALB / c mice were purchased from Charles River Laboratories. Fig. 2E depicts the preparation of Poly(2-ethyl-2-oxazoline), alkyne terminated 24 with p- Toluenesulfonyl chloride 23 according to a preferred embodiment.

[0109] Poly(2-ethyl-2-oxazoline) (0.500 g, 0.100 mmol) 24 was added into a round-bottom flask, followed by 20 mL of dry acetonitrile. The acetonitrile was then evaporated under reduced pressure and reintroduced thrice to eliminate residual water from the poly(2-ethyl-2-oxazoline) 24. Subsequently, 20 mL of dichloromethane (DCM) 36 was added to the flask containing the dry Poly(2-ethyl-2-oxazoline) 24, and the mixture was stirred in an ice bath for 30 minutes. Following this, triethylamine (0.050 g, 0.500 mmol) 34 was added, and i-toluenesulfonyl chloride (0.475 g, 2.50 mmol) 23 was slowly added dropwise over three hours under ice-cold conditions. The solution was stirred at room temperature for 24 hours and then subjected to a temperature increase to 37 °C for the subsequent 24 hours. The solution was extracted with 10% Citric acid and then washed with brine and distilled water. The organic layer was evaporated under reduced pressure to a minimal volume of DCM 36 and then precipitated three times in diethyl ether (Et2O). The product was obtained as a slightly yellow sticky material (0.370 g, 71.7%).!H NMR (600 MHz, CDCh) 87.69 (d, J= 8.3 Hz, 3H), 7.28 (d, J= 8.0 Hz, 3H), 4.12 (d, J= 7.2 Hz, 2H), 3.44 (m, J = 24.1, 10.7 Hz, 187H), 3.14 (d, J= 7.2 Hz, 1H), 2.45 - 2.25 (m, 99H), 1.12 (m, J= 18.9, 15.0, 7.1 Hz, 147H).

[0110] Fig. 2F depicts a further detailed view of the formation of DE-POZ 32 reaction of Fig. 2B according to a preferred embodiment. G4-PAMAM dendrimer (0.050 g, 0.003 mmol) 21, POZ- OTs (0.180 g, 0.035 mmol) 25, Triethylamine (0.003 g, 0.035 mmol) 34, and 20 mL of ethanol (EtOH) were added in a round bottom flask and were left to stir for five days under N2 for five days at 37 °C. The solution was then passed through a centrifugal filter with a cutoff 10 kDa. After several washings, the upper solution was left to dialyze with a dialysis membrane 8 kDa cut off overnight against ethanol. The desired solution was evaporated under reduced pressure and was obtained as brown to yellow sticky material (0.160 g).

[0111] Figs. 2G - 21 depict steps for formation of the peptide 16 to be used in the formation of DE-POZ -PE 30.

[0112] Fig. 2G depicts synthesis of palmitoyl pentapeptide-4-Boc 29. Palmitoyl 4-pentapeptide (0.500 g 0.623 mmol) 26, triethylamine (0.180 g, 1.24 mmol), 34 and 100 mL dimethylformamide (DMF) 40 were added to a round bottom flask and tertbutyloxycarbonyl (0.270g, 1.24 mmol) dropwise over two hours under ice-cold conditions. The solution was left to stir at room temperature, loosely capped overnight. The solvent was evaporated under reduced pressure and then precipitated three times in acetonitrile (ACN). The product was obtained as a white powder (0.460 g, 72.2%).!H NMR (600 MHz, DMSO) 5 5.10 (s, 1H), 4.98 (s, 1H), 4.29 (m, 2H), 4.19 (m, J = 22.2 Hz, 2H), 4.03 (m, 1H), 3.71 - 3.58 (m, 4H), 2.85 (t, 4H), 2.16 - 2.06 (t, 2H), 1.79 - 1.57 (m, 4H), 1.54 - 1.42 (m, 6H), 1.35 (s, 18H), 1.22 (m, 28H), 1.07 - 0.97 (t, 6H), 0.84 (t, J= 6.9 Hz, 3H).

[0113] Fig. 2H depicts synthesis of 5-azido pentanol 44.

[0114] 5 -Bromo- 1 -pentanol (0.500 g, 2.99 mmol) 42, sodium azide (0.380 g 5.98 mmol) 38, and 50 mL of dimethylformamide (DMF) 40 were added in a round bottom flask and left to stir at 80 °C overnight. The solvent was removed under reduced pressure, and the oily crude was dissolved in diethyl ether (Et2O) and was washed with brine and water. The organic layer was removed under reduced pressure, and the product was obtained as a colorless oily product (0.350 g, 92%).JH NMR (600 MHz, CDCh) 5 3.61 (m, J = 11.2 Hz, 2H), 3.25 (t, J = 13.8 Hz, 2H), 1.64 - 1.53 (m, 4H), 1.42 (m, J= 7.6 Hz, 2H).

[0115] Fig. 21 synthesis of palmitoyl pentapeptide-4-Boc-ester-azide 46.

[0116] Palmitoyl pentapeptide-4-Boc (0.300 g, 0.300 mmol) 29, l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide( EDC HC1) (0.093 g, 0.600 mmol) and 30 mL of dimethylformamide (DMF) 40 were added in a round bottom flask and were left to stir together for 10 minutes under N2 and also under ice-cold conditions. Then N,N-Diisopropylethylamine (DIPEA) (0.070 g, 0.600 mmol) were added to the above solution and stirred together for 10 minutes under ice-cold conditions. Finally, 5-azido pentanol (0.056 g, 0.450 mmol) 44 was added, and the solution was left to stir under ice-cold conditions for 1 hour and then switched to room temperature overnight (0.280 g, 84%). 'H NMR (600 MHz, DMSO) 5 5.02 (s, J= 64.6 Hz, 2H), 4.23 (m, J= 33.7 Hz, 4H), 4.01 (m, 3H), 2.89 - 2.80 (t, 4H), 2.15 - 2.06 (t, 2H), 1.63 (m, 4H), 1.47 (m, 10H), 1.35 (s, 18H), 1.22 (m, 32H), 1.08 - 0.99 (t, 6H), 0.84 (t, J= 6.9 Hz, 3H).

[0117] Fig. 2J depicts synthesis of DE-POZ-PE 30. DE-POZ (0.155 g) 32, CuSO4 (0.070 g, 0.280 mmol), sodium ascorbate (0.167 g, 0.840 mmol), Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (0.050 g, 0.094 mmol), palmitoyl pentapeptide-4-Boc-ester-azide (0.256 g, 0.280 mmol) 46 and 10 mL of water was stirred at room temperature for 12 hours. The solution was then passed through a centrifugal fdter with a cutoff 10 kDa. After several washings with water and 0.500 M EDTA, the upper solution was left to dialyze with a dialysis membrane 8 kDa cut off overnight against 0.500 M EDTA for 3 hours and then water overnight. The desired solution was evaporated under reduced pressure and was obtained as light brown sticky material (0.300 g). Afterward, the material was redissolved again in a solution comprising 10 mL of trifluoroacetic acid (TFA) and di chloromethane (DCM) 36 at a ratio of 1 : 1. Following a 10-hour stirring period at room temperature, the solvent evaporated through rotary evaporation. The samples were dialyzed against distilled water, and the product was obtained as light-yellow sticky material (0.200 g).

[0118] Fig. 2K depicts synthesis of 3 -azido-propanol 52.

[0119] 3 -Bromo- 1 -propanol (0.397 g, 2.86 mmol) 50, sodium azide (0.371 g 5.72 mmol), and 20 mL of DI water were added in a round bottom flask and stirred at 90 °C overnight. The reaction was allowed to cool and then quenched with a cooled 15.5 M KOH (10 mL) solution. The solution was extracted three times with diethyl ether. The organic layer was collected and dried over magnesium sulfate. After fdtration, the solvent was removed under reduced pressure, and the product was obtained as a slightly yellow colorless oil (0.277 g, 96%). 1H NMR (600 MHz, CDC13) 5 3.62 - 3.48 (m, 2H), 3.37 - 3.22 (m, 2H), 1.79 - 1.61 (m, 2H).

[0120] Fig. 2L depicts synthesis of Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA).

[0121] To a round bottom flask (100 mL) tripropargylamine (0.266 g, 2.03 mmol) 54, 3-azido propanol (0.821 g, 8.10 mmol) 52, 2,6-lutidine (0.217 mmol, 2.03 mmol) 56, and 10 mL of a 1 : 1 acetonitrile:MeOH mixture were added under cooled conditions and left to stir for 15 minutes under N2. Then Cu(CH3CN)4BF4 (0.017 g, 0.060 mmol) was added, and the solution was left to stir for 3 d under N2at room temperature. The solvent was then removed under reduced pressure, and the crude was redissolved in minimal methanol. Then, the solution was added slowly in cooled diethyl ether, and precipitates were observed. The product was collected as a white solid (0.440 g, 50%). 'HNMR (600 MHz, D2O) 5 7.94 (s, 3H), 4.48 (t, J= 7.1 Hz, 6H), 3.80 (s, 6H), 3.54 (m, J= 6.1 Hz, 6H), 2.10 (m, J= 6.8 Hz, 6H).

[0122] Experimental Results

[0123] Fig. 3 A depicts Matrix-Assisted Laser Desorption / Ionization Time of Flight (MALDI- TOF) of DE-POZ 32 in accordance with the principles of the present invention. To confirm the molecular weight of DE-POZ 32 and DE-POZ-PE 30, mass spectrometric analysis was conducted on dendrimers 21 using a MALDI-TOF (matrix-assisted laser desorption / ionization- time of flight) instrument. The instrument operated in positive ion reflector mode, employing a pulsed nitrogen laser (337 nm) with a 19 kV acceleration voltage. The matrix used for the analysis was 2',4',6'-Trihydroxyacetophenone monohydrate (THAP).

[0124] Fig. 3B depicts MALDI-TOF of DE-POZ-PE 30 in accordance with the principles of the present invention.

[0125] Fig. 3C depicts Dynamic Light Scattering Spectroscopy (DLS) of DE 21, DE-POZ 32, and DE-POZ-PE 30 in accordance with the principles of the present invention. Measurements were conducted on a Malvern Zetasizer Nano ZS at 25 °C. All samples were measured after final purification in a 1.5 mL cuvette with a 633 nm laser, 175 scattering angle, and a medium refractive index of 1.51.

[0126] Figure 3D depicts *H NMR of Poly(2-ethyl-2-oxazoline)-tosylchloride 25 as obtained by the procedure herein.

[0127] Figure 3E depicts1H NMR of palmitoyl pentapeptide-4-Boc 29 as obtained by the procedure herein.

[0128] Figure 3F depicts 'H NMR of 5-azido pentanol 44 as obtained by the procedure herein.

[0129] Figure 3G depicts *H NMR of palmitoyl pentapeptide-4-Boc-ester-azide 46 as obtained by the procedure herein.

[0130] Figure 3H depicts ES MS of palmitoyl pentapeptide-4-Boc-ester-azide 46. To confirm the molecular weight of palmitoyl pentapeptide-4-Boc-ester-azide 46, an LC / ESLMS system composed of an Agilent 1 100 series HPLC system followed by a 4000 QTRAP mass spectrometer was used. HPLC was performed using a reverse-phase Kinetex 2.6 pm XB-C18 50x 2.1 mm column with a gradient elution of 10-90% acetonitrile in water for 8 minutes, 210 nm wavelength, injection volume 4 pl, and flow 0.4 mL / min.

[0131] Figure 31 depicts 'H NMR of 3 -azido-propanol 52 as obtained by the procedure herein.

[0132] Figure 3 J depicts ’HNMR of Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) 58 as obtained by the procedure herein.

[0133] Release studies

[0134] Fig. 4A depicts a visual representation of conduction of the release study experiments in accordance with the principles of the present invention. To assess the drug release from all three formulations, such as DE 21, DE-POZ 32, and DE-POZ-PE 30, 30 mg of retinol 28 was loaded in each formulation. After the loading efficiency was calculated as above, release studies were started. The all-trans retinol-dendrimer complexes were dissolved in 1 mL 0.1 M potassium phosphate buffer with 10% methanol at an appropriate pH (5 or 7.4). Then, the dialysis bag was submerged in 30 mL 0.1 M potassium phosphate buffer with 10% methanol at appropriate pH (5 or 7.4) and a temperature of 25 °C and 37 °C. Methanol was used to mimic the oils in the skin. The outer phase was stirred for 24 hours, and 100 pL of solution was withdrawn at specific time intervals (1, 2, 4, 6, 8, 18, and 24 h). The amount of all-trans retinol 28 in the samples withdrawn from the outer phase over a 24-h period was determined by UV-Vis at 325 nm and then was added back to the outer phase. The values were then plotted in a graph.

[0135] Dendrimers 12 are characterized by three main parts: (1) a central core 12a, (2) the repeated units 12b that grow into branches and determine the microenvironment of the dendrimer interior, and lastly, (3) the dendrimer terminal groups 12c exposed at the surface.

[0030]

[0136] Dendrimers 12 enhance the solubility of poorly water-soluble molecules by encapsulating them in the interior cavity using non-covalent interactions. [17, 31] Despite having a hydrophobic core, the whole composite is water-soluble because of the surface polar groups that interact with water, granting dendrimers 12 the ability to dissipate in aqueous solutions.

[0137] The encapsulation of a hydrophobic active ingredient, all-trans retinol 28, as assessed and studied its release rates under varying conditions.

[0138] The pH of the skin is acidic, ranging from a pH of 4.0 at the upper layers of the epidermis to 6.5. Skin also ranges in temperature from about room temperature to body core temperature of 37°C, depending on the depth.

[0139] Initially, all-trans retinol 28 was loaded into specific systems, including DE 21, DE-POZ 32, and DE-POZ-PE 30, through a 12-hour incubation in methanol under inert conditions with slow stirring (Figure 4A (1)).

[0140] The non-encapsulated all-trans retinol 28 was removed by using ultra centrifugal filters with molecular cut of 3.5kDa (Figure 4A (2)).

[0141] The loading of all-trans retinol 28 was higher in DE-POZ-PE 30 (83%), then in DE-POZ 32 (62%), and the lowest in DE 21 (58%) (Figure 5). These percentages were calculated by quantifying the amount of retinol in the nanocarrier and the free retinol in the supernatant (or filtrate) using UV-Visible spectroscopy.

[0142] Amount of Retinol in Nanocarrier=Total Retinol Added-Free Retinol

[0143] Loading efficiency: Amount of Retinol in Nanocarrier / total amount of retinol added

[0144] The in vitro release behavior of all-trans retinol 28 from its dendrimer systems (DE 21, DE-POZ 32 and DE-POZ-PE 30) was investigated using a dialysis membrane with a molecular cut of 3.5 kDa. The all-trans retinol-dendrimer complexes were dissolved in 1 m 0.1 M potassium phosphate buffer with 10% methanol in it at appropriate pH (5 or 7.4) (Figure 4A (3)).

[0145] Then the dialysis bag was submerged in 30 mL 0.1 M potassium phosphate buffer with 10% methanol in it at appropriate pH (5 or 7.4) and temperature 25 °C and 37 °C (Figure 4A (3)). The outer phase was stirred for 24 hours and, at the appropriate time (1 , 2, 4, 6,8, 18 and 24h) was withdrawn ImL which was directly substituted with 1 mL of fresh dissolution medium (Figure 4 A (4)).

[0146] The amount of all-trans retinol 28 in the samples withdrawn from outer phase over a 24-h period was determined by UV-Vis.

[0147] The absorbance at 325 nm was recorded and by Beer-Lambert law the concentration of all-trans retinol was calculated (Figure 4A (5)).

[0148] The system is pH and temperature-sensitive (Figure 4).

[0149] The pH sensitivity is a feature that results of the presence of tertiary amines in the core of dendrimers 21. These tertiary amines have a pKa of 6.3, and at low pH, they are susceptible to protonation, thereby interacting more strongly with water molecules. [32-34]

[0150] Therefore, the environment inside the dendrimer core 21a becomes more prone to accept the water in its cavities.

[0151] In this moment, the active ingredient in the core 21a is allowed to diffuse from the dendrimer 21 into the solution.

[0152] In addition, at low temperatures the system is less hydrated, causing dendrimers 21 to form a shell that prevents active ingredients from escaping its core 21a.

[0035]

[0153] In general, biologically active ingredients are used to improve skin health, however it is shown that they also cause local skin irritation and peeling.

[0036]

[0154] One of the factors might be burst release of active ingredients in the skin.

[0155] For this reason, a system that can slowly release the active ingredient in the skin is highly needed.

[0156] All-trans-retinol 28 releases rate from G4-PAMAM dendrimer (DE) 21 for around 8 hours (Figure 4B). However, the release is extended up to 24 hours when the delivery system is DE-POZ 32 (Figure 4C) and DE-POZ-PE 30 (Figure 4D).

[0157] Incorporating the peptide in the system (DE-POZ-PE) 30 does not necessarily elevate the release rate compared to DE-POZ 32 because of the hydrolyzation of ester bond which connects the peptide to system (Figure 4D).

[0158] Many skincare products that people use are usually stored at room temperature. To mimic those conditions, a release study at room temperature (25 °C) and pH 7.4 was performed. No active ingredient was released from DE 21 (Figure 4B), DE-POZ 32 (Figure 4C) and DE-POZ- PE 30 (Figure 4D) under these conditions.

[0159] Fig. 4B depicts a release of retinol 28 from DE 21 in accordance with the principles of the present invention. As shown, the all-trans retinol 28 was 100% released within 8 hours at pH 5 and 37°C. Very little all-trans retinol 28 was released at pH 7.5 and 25°C over 24 hours.

[0160] Fig. 4C depicts a release of retinol 28 from DE-POZ 32 in accordance with the principles of the present invention. As shown, the all-trans retinol 28 gradually released over 24 hours at pH 5 and 37°C. Very little all-trans retinol 28 was released at pH 7.5 and 25°C over 24 hours.

[0161] Fig. 4D depicts a release of retinol 28 from DE-POZ-PE 30 in accordance with the principles of the present invention. As shown, the all-trans retinol 28 gradually released over 24 hours at pH 5 and 37°C. Very little all-trans retinol 28 was released at pH 7.5 and 25°C over 24 hours.

[0162] Fig. 5 depicts the loading efficiency of all-trans retinol 28 in three different systems: DE 21, DE-POZ 32, and DE-POZ-PE 30.

[0163] For purposes of illustration, the loading experiment was conducted by incubating 30 mg of all-trans-retinol 28 with 30 mg of DE 21, DE-POZ 32, or DE-POZ-PE 30 in methanol for 12h with constant slow stirring. Next, the solution was passed through a centrifugal filter with a cut of 3.5kDa. The solution was constantly washed until no free all-trans retinol 28 was observed in the filtrate from the centrifugal filter. The filtrate was collected, and the concentration of unloaded all-trans-retinol 28 was determined by UV-Vis. The amount of loaded all-trans-retinol 28 was then calculated by subtracting the amount of unloaded all-trans-retinol 28 from the initial 30 mg used in the experiment. According to the preferred embodiment, it is found that the loading of all-trans retinol 28 was higher in DE-POZ-PE 30 (83%), than in DE-POZ 32 (62%), and the lowest in DE 21 (58%).

[0164] Cell Viability

[0165] Primary Dermal Fibroblast: Normal, Human, Adult (HDFa) cells were grown in Fibroblast Basal Medium supplemented with Fibroblast Growth Kit-Serum-free. The cells were seeded at a concentration of 2 x 104 cells / mL in a 96-well MatTek culture slide (100 pL / well) and allowed to adhere overnight in a 37 °C, 5% CO2 incubator. The following day, the cells were treated in triplicates with different concentrations of DE 21, DE-POZ 32, and DE-POZ-PE 30 for 4 hours and 24 hours. Subsequently, cytotoxicity was detected using a lactate dehydrogenase (LDH) assay.

[0166] Cytotoxicity

[0167] The cytotoxicity of the dendrimer and dendrimer complexes was tested, with results presented in Figs. 6A - 6C. Fig. 6A depicts a graph of the cell viability of DE 21 in accordance with the principles of the present invention. Fig. 6B depicts a graph of the cell viability of DE- POZ 32 in accordance with the principles of the present invention. Fig. 6C depicts a graph of the cell viability of DE-POZ-PE 30 in accordance with the principles of the present invention.

[0168] G4-PAMAM dendrimers 21 are known for their cytotoxicity due to their positively charged primary amine, which can strongly interact with the negatively charged cell membrane. One possible solution involves reducing the surface charge of dendrimers by covalently attaching a polymer which could hinder the charges to some degree.

[0169] Fig. 6A shows that the cytotoxicity of G4-PAMAM dendrimer (DE) 21 increases once the concentration is increased up to 0.1 mg / mL. However, the cytotoxicity of G4-PAMAM dendrimer 21 decreased significantly once modified with polyoxazoline 24 (Figure 6B) and peptide 16 (Figure 6C). Skin penetration studies

[0170] Fig. 7A depicts a skin penetration study on naive porcine skin imitating human skin texture in accordance with the principles of the present invention. Fig. 7B depicts a skin penetration study on porcine skin incubated Cy5 in accordance with the principles of the present invention. Fig. 7C depicts a skin penetration study on porcine skin incubated with DE(Cy5) in accordance with the principles of the present invention. Fig. 7D depicts a skin penetration study on porcine skin incubated with DE-POZ(Cy5) in accordance with the principles of the present invention. Fig. 7E depicts a skin penetration study on porcine skin incubated with DE-POZ- PE(Cy5) in accordance with the principles of the present invention.

[0171] One of the other reasons for skin irritation caused by active ingredients might be accumulation of the formulation on the epidermis surface 8 for a long time. It is discovered that accumulation is mainly caused by the inability of the delivery system to deliver retinol 28 deep in the dermis layer 6 of the skin and / or penetrate the epidermis 8. Therefore, embodiments of the present invention provide for adequate penetration of the epidermis 8.

[0172] The reason why delivery of active ingredients in the dermis 6 is important is because in the dermis 6 the extracellular matrix is found, where the fibroblast cells are located. Fibroblast cells are the main cells that produce collagen and elastin. Thereby any active ingredient which helps fibroblast to produce more collagen and elastin needs to reach the dermis 6 to completely do its function. To prove that the modified system could penetrate the epidermis and reach the dermis, an ex-vivo model such a porcine skin was used (Figure 7).

[0173] The skin was equilibrated between the donor and acceptor chamber of Franz diffusion cells. Prior to skin incubation Cy5 was loaded in each sample: DE 21, DE-POZ 32, DE-POZ-PE 30. Thus, DE-Cy5, DE-POZ-Cy5 and DE-POZ-PE were added respectively in the donor chamber of Franz diffusion cell, above the epidermis facing skin. The above system was left for 12h at 37°C. Afterward, the skin was processed, cryosectioned, and imaged.

[0174] Cy5 was incubated under identical conditions, and no notable fluorescence was observed in the skin, likely attributed to tissue processing that occurred before cryosectioning, potentially leading to washout (Figure 7B). The results show that DE-Cy5 is mostly located in the epidermis part 8 of the skin (Figure 7C). However DE-POZ reaches the dermis 6 (Figure 7D), and DE-POZ-PE is present way more in dermis 6, possibly owning the peptide feature (Figure 7E). The fatty acid chain in the peptide helps the delivery system to penetrate better the skin, by interacting with the lipid bilayer of skin.

[0175] An example according to a preferred embodiment is described herein. Freshly excised porcine skin was gently washed with a 1% (v / v) liquid detergent solution. The skin was then cut to a diameter of 20 mm by a biopsy punch. The skin biopsy was placed epidermis-side up between the donor and receiver chambers of a Franz diffusion cell. The skin surface temperature was maintained at 37 °C using a heated circulating water bath. Dendrimer complexes (1 mg) were loaded with 0.010 mg of Cy5, which resulted in 100% encapsulation for all formulations. All the formulations were then applied to the skin in aqueous solutions of 50 pL for 12 hours. After 12 hours, the surface of the skin was washed gently with a diluted detergent solution and rinsed twice with 2 m MilliQ water to remove any unabsorbed Cy5 in the skin. The excess liquid from the skin was removed by dabbing it in cotton swabs. Later, the skin disks were incubated under shaking conditions in a solution with 10% paraformaldehyde (PF A) in IX PBS for 48 hours. Skins were then embedded with O.C.T. compound in the cryo molds under liquid nitrogen and further cryo-sectioned with LEICA CM 1860 into 10 mm thick and collected on positively charged slides. Once dried, slides were dipped in 10% PFA (10 min) and DAPI (1 :2000). Lastly, to wash the excessive dye, the slides were dipped 2 times for 20 min each in 0.100 M PBS solution pH 7.40. The skin sections were then visualized using epi-fluorescent microscopy under DAPI and Cy7 channels to determine the penetration of fluorescent dye in the skin layers.

[0176] Histological analysis of organs

[0177] Another example according to a preferred embodiment is described herein. BALB / c mice, 6-8 weeks old were anesthetized using isoflurane chamber and then exactly 0.100 mg of DE 21, DE-POZ 32, and DE-POZ-PE 30 was topically applied on the shaved skin. Twenty-four hours post-application, mice were sacrificed, and their skin was collected. Each skin was then left for 30 min with Phosphate-buffered saline (PBS) under shaking conditions. Next, the skins were transferred to 30 ml of 4% Paraformaldehyde (PFA) in 1 x PBS again under shaking conditions for 48 hours. Skins were then embedded with O.C.T. compound in the cryo molds under liquid nitrogen and further cryo-sectioned with LEICA CM 1860 into 10 mm thick and collected on positively charged slides. Once dried, slides were dipped in (i) 10% PFA (10 min), (ii) lx PBS (5 min), (iii) hematoxylin (1.50 min), (iv) tap water (10 dunks), (vi) tap water (10 dunks), (vii) tap water (10 dunks), (viii) accumate ( 0.25% Acid Alcohol) (4 dunks), (ix) tap water (10 dunks), (x) Scott's tap water (1 min), (xi) 95% ethanol (30 sec), (xii) eosin 1% acetic acid (1 min), (xiii) 95% ethanol (10 dunks), (xiv) 100% ethanol (1 min), (xv) 100% ethanol (1 min), (xvi) xylenes (1 min). The slides containing the stained samples were then cleaned and sealed using CYTOSEAL 60 and a cover slip. Lastly, the slides were imaged under a brightfield microscope.

[0178] Fig. 8A-E depicts colorized Fig. 7A-E.

[0179] Fig. 9 depicts a graph of human dermal fibroblasts incubated with different treatment, such as, cells only without any stimulants and cells with stimulants, such as, TGF-pi and ascorbic acid which stimulate collagen production. Next all-trans retinol (RE) 28, DE-POZ(RE), PE and DE-POZ-PE 30. After incubation cells were stained with picro-sirius red dye to visualize collagen production intensity in each sample in accordance with the principles of the present invention.

[0180] Collagen stimulation assay

[0181] To demonstrate the ability of the system according to embodiments of the present invention to promote collagen production, Primary Dermal Fibroblast; Normal, Human, Adult (HDFa) cells were used. Initially, cells were incubated with only the cell media (Cells only), followed by incubation with cell media supplemented with conventional stimulants like TGF-01 and ascorbic acid (Stimulated). Additionally, separate samples were incubated with palmitoyl pentapeptide 26 alone (PE), and DE-POZ-PE 30 which was adjusted to contain a comparable amount of surface-decorated peptide. For consistency, an equal quantity of all-trans retinol 28 was subsequently loaded into DE-POZ-PE 30 (DE-POZ-PE(RE)), matching the amount present in the sample containing only all-trans retinol (RE) 28.

[0182] Primary Dermal Fibroblast; Normal, Human, Adult (HDFa), available at ATCC product code: PCS-201-030, were plated at a seeded at 9 * 103cells into 24 well tissue plates (n=4) in Fibroblast Basal Medium, available at ATCC product code: PCS-201 -030, supplemented with 5% FBS and 5 pg / mL Insulin. Cells were allowed to adhere overnight, and then the next day, the media was removed, and cells were washed with phosphate-buffered saline (PBS) and replaced with samples such as RE (0.01 pg / mL), DE-POZ-PE(RE) (0.13 mg), PE (0.008mg) and DE- POZ-PE (0.13 mg). Negative control (fibroblast basal media only) and positive control (ascorbic acid 50 pg / mL and rh EGF / TGF P-1 Supplement 5 ng / mL 30 pg / mL and rh insulin 5 pg / mL) were used. Cells were incubated for 3 days. Positive controls were added as they are known to stimulate type I and III collagen. After incubating cells in culture for 3 days, the culture media was aspirated, and the cells were treated with 70% ice-cold ethanol to fix any deposited material onto the surface of the tissue culture plastic. After fixation, the samples were promptly transferred to a -80 °C freezer for 10 minutes to ensure complete fixation. Subsequently, the ethanol solution was decanted, and each well was gently rinsed with distilled water. The cells were then exposed to a solution containing Sirius Red and picric acid and incubated overnight at 4 °C with gentle agitation. The next day, the excess dye was removed by rinsing the fixed cells with distilled water multiple times until residual unbound dye and excess water were eliminated. The cells were treated with 1 M NaOH at room temperature for 10 min with gentle agitation to dissolve the collagen dye complex and ensure thorough mixing. Following this, 100 pL aliquots of each sample were transferred in duplicate to a 96-well microplate, and the absorbance was measured at 490 nm using a microplate reader.

[0183] After staining cells with picro-sirius red dye to visualize collagen production, it was observed that cells incubated with DE-POZ-PE 30 produce more collagen. This is attributed to possible more efficient transport of the peptide in the cells compared to PE sample. As expected, when cells were incubated with DE-POZ-PE(RE) the collagen stimulation is higher due to both collagen and all-trans retinol stimulate collagen production.

[0184] Conclusion

[0185] Embodiments of the present invention include a nanocarrier which is pH and temperature sensitive and can enhance skin penetration into the dermal layer of the skin. Further embodiments of the present invention include complexes of combined G4-PAMAM dendrimers 21, which are known for their small size, with polyoxazolines 24, which enhance solubility and decrease surface charge. In addition, the dendrimer-polyoxazolines 32 complex was then grafted with palmitoyl pentapeptide 27 which resulted in DE-POZ-PE 30, having enhanced skin penetration and stimulated collagen and elastin production.

[0186] DE-POZ-PE 30 demonstrates slow-release rate of active ingredient at pH 5 and 37°C, however no release was observed at pH 7.4, 25°C.

[0187] The cytotoxicity of G4-PAMAM dendrimers 21 was also reduced by coating them with polyoxazoline 24 and peptide 27.

[0188] DE-POZ-PE nanocarrier 30 can penetrate the skin and reach the desired layer, the dermis 6. That feature is possibly attributed to the size and the presence of palmitoyl pentapeptide 27 in the surface of the nanoparticle.

[0189] The full system (DE-POZ-PE) 30 is able to stimulate collagen once incubated with cells. This is attributed to possible better cell trafficking of peptide when it is attached to DE-POZ 32.

[0190] Incorporation Procedure for DE-POZ-PE in Topical Cream / Lotion / Oil / Serum Products

[0191] By following these steps, the active ingredient loaded DE-POZ-PE 30 can be effectively incorporated into a topical product, resulting in a stable and homogeneous formulation.

[0192] To incorporate the activated dendrimer complex into a topical product such as sunscreen, cream, lotion, oil, or serum using a homogenizer, the following systematic process should be followed to ensure homogeneity and stability:

[0193] • Preparation: Measure and prepare the activated dendrimer powders 12 at a concentration ranging from 0.001% to 12.0% of the final product.

[0194] • Initial Mixing: Slowly add the activated dendrimer powders to the bulk cream, serum, or other topical base with gentle mixing.

[0195] • Homogenization: Perform continuous stirring or mixing with a homogenizer set at 4000 to 15,000 RPM, maintaining a temperature range of 0 to 40°C (avoid temperatures exceeding 40°C). This step ensures the even distribution of the dendrimer throughout the product. Continue homogenizing until uniformity is achieved, ensuring that the dendrimer powders are thoroughly incorporated into the product. This process may require periodic adjustments to mixing speed and duration to achieve optimal results. • Cooling and Filtration: If the product was heated during mixing, allow it to cool to room temperature. Filter and transfer the homogenized product into clean, sterile containers with a 10- 100pm pore size mesh filter.

[0196] • Packaging: Seal and label the containers appropriately, ensuring the product is ready for distribution or further testing.

[0197] Unless otherwise specified herein, materials identified above have been purchased or are purchasable from Sigma Aldrich.

[0198] Although the invention has been discussed with reference to specific embodiments, it is apparent and should be understood that the concept can be otherwise embodied to achieve the advantages discussed. In this regard, the foregoing description of the systems and methods is presented for purposes of illustration and description.

[0199] Furthermore, the description is not intended to limit the invention to the form disclosed herein. Accordingly, variants and modifications consistent with the following teachings, skill, and knowledge of the relevant art, are within the scope of the present invention. The embodiments described herein are further intended to explain modes known for practicing the invention disclosed herewith and to enable others skilled in the art to utilize the invention in equivalent, or alternative embodiments and with various modifications considered necessary by the particular application(s) or use(s) of the present invention.

[0200] References

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Claims

CLAIMSWhat is claimed is:

1. A modified nanoparticle nanocarrier system for delivering one or more active ingredients into a skin comprising: a dendrimer; a linker; and a peptide.

2. The modified nanoparticle nanocarrier system of claim 1, wherein the dendrimer is G4- polyamidoamine (PAMAM) dendrimer.

3. The modified nanoparticle nanocarrier system of claim 1, wherein the linker is polyoxazoline (POZ).

4. The modified nanoparticle nanocarrier system of claim 1, wherein the peptide is palmitoyl pentapeptide.

5. The modified nanoparticle nanocarrier system of claim 1 further comprising one or more active ingredients.

6. The modified nanoparticle nanocarrier system of claim 1, wherein the dendrimer is first coated with the linker, wherein the linker further comprises a leaving group to form a dendrimer-linker conjugate.

7. The modified nanoparticle nanocarrier system of claim 6, wherein the dendrimer-linker conjugate is coupled to the peptide to form a dendrimer-linker-peptide conjugate.

8. The modified nanoparticle nanocarrier system of claim 1, wherein the peptide is an enzymatically cleavable peptide.

9. A method of synthesizing a modified nanoparticle nanocarrier for delivering one or more active ingredients into a skin comprising: coating a dendrimer with a linker having a leaving group to form a dendrimer-linker conjugate; coupling the dendrimer-linker-conjugate to a peptide.

10. The method of synthesizing a modified nanoparticle nanocarrier of claim 9, further comprising modifying the peptide to form an enzymatically cleavable peptide.

11. The method of synthesizing a modified nanoparticle nanocarrier of claim 9, wherein the dendrimer is G4-polyamidoamine (PAMAM) dendrimer.

12. The method of synthesizing a modified nanoparticle nanocarrier of claim 9, wherein the linker is polyoxazoline (POZ).

13. The method of synthesizing a modified nanoparticle nanocarrier of claim 9, wherein the peptide is palmitoyl pentapeptide.

14. The method of synthesizing a modified nanoparticle nanocarrier of claim 9, further comprising loading the modified nanoparticle nanocarrier with one or more active ingredients.

15. A method of delivering one or more active ingredients into the skin, the method comprising the steps of: synthesizing a dendrimer-linker conjugate by coating a dendrimer with a linker having a leaving group; synthesizing a dendrimer-linker-peptide conjugate by coupling the dendrimer-linker conjugate to a peptide to form a modified nanoparticle nanocarrier; loading the modified nanoparticle nanocarrier with one or more active ingredients to form an activated nanocarrier; measuring and preparing the activated nanocarrier; adding the activated nanocarrier to a topical base; mixing the activated nanocarrier with the topical base to achieve a homogenized product; cooling and filtering the homogenized product; and applying the homogenized product onto the skin.

16. The method of delivering one or more active ingredients into the skin of claim 15, wherein the dendrimer is G4-polyamidoamine (PAMAM) dendrimer.

17. The method of delivering one or more active ingredients into the skin of claim 15, wherein the linker is polyoxazoline (POZ).

18. The method of delivering one or more active ingredients into the skin of claim 15, wherein the peptide is palmitoyl pentapeptide.

19. The method of delivering one or more active ingredients into the skin of claim 15, wherein the peptide is an enzymatically cleavable peptide.

20. The method of delivering one or more active ingredients into the skin of claim 15, wherein the topical base is a topical cream, a topical lotion, a topical oil, or a topical serum.

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