Bioactive growth factors for dermal topical applications

Abstract

Disclosed are novel formulations for dermal topical applications and methods for making the same. The formulations comprise oleosome-emulsified fibroblast growth factor 2 (FGF-2). The formulations may be used to treat skin conditions, including conditions associated with skin aging and skin injury.

Description

TITLE: BIOACTIVE GROWTH FACTORS FOR DERMAL TOPICAL APPLICATIONS RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Application No. 63 / 677,107 filed July 30, 2024; the entire contents of United States Patent Application Nos.63 / 677,107 is hereby incorporated by reference. INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “P96520222PCT00_ 96520222_SequenceListing.xml” (36,308 bytes), filed herewith by electronic submission and created on July 8, 2025, is herein incorporated by reference. FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to dermal topically applied formulations, and notably to dermal topical formulations comprising growth factors. BACKGROUND OF THE DISCLOSURE

[0004] The following paragraphs are provided by way of background to the present disclosure. They are not however an admission that anything discussed therein is prior art or part of the knowledge of a person of skill in the art.

[0005] Dermal topical application of bioactive substances, such as growth factors, can offer a range of health benefits. Thus, for example, topically applied bioactives can exert anti-oxidative, anti-inflammatory, wrinkle-reducing, and anti-aging effects, as well as promote hair growth, and enhance the structure and integrity of the skin. Desired outcomes from dermal topical application of bioactive substances include, for example, the visible reduction of aging signs, such as spots, wrinkles, pigmentation, loss of skin tone, rough, uneven skin texture, blood flow (broken capillaries), redness, and blotchiness. In cases where the skin barrier is compromised due to injury, the topical administration of bioactive substances, such as growth factors, can significantlyaccelerate the healing of acute wounds, including superficial burn injuries, deep burn injuries, trauma, and surgical incisions. Additionally, these bioactive substances can be used to manage various dermatological conditions, such as alopecia, psoriasis, atopic dermatitis, acne, and diabetic foot ulcers, for example.

[0006] Bioactive substances can be incorporated in a variety of dermal topical formulations including gels, creams, crèmes, lotions, serums powders, and the like. In general, these formulations must be designed to effectively penetrate the skin’s natural barrier structures, such as biological membranes and the skin’s outer tissue layer, known as stratum corneum, in order to deliver the bioactives to their intended target sites within the epidermis and / or dermal tissue layers of the skin. Furthermore, the formulation must preserve the biological activity of the bioactive substance both within the product, and upon application to the skin, to ensure therapeutic efficacy.

[0007] Growth factors in humans are naturally secreted by various skin cell types within the epidermis and dermis, including keratinocytes, fibroblasts, and melanocytes. These factors regulate cell growth, proliferation, and differentiation, playing a critical role in maintaining healthy skin structure and function. While growth factors can trigger multiple cellular responses, their primary function in skin care is to stimulate collagen and elastin production, enhancing skin firmness and elasticity, through promotion of gene expression and cellular proliferation.

[0008] The effectiveness of dermal topically applied growth factors substantially depends on their stability and ability to penetrate the skin barrier, notably the stratum corneum, to reach target tissues. In this respect, fibroblast growth factor 2 (FGF-2) is particularly unstable in solution and highly susceptible to degradation. Thus, for example, FGF-2 is thermally unstable and loses activity over time. Even at 4 °C , reconstituted solutions of FGF-2 can be stable for only a few hours (Benington, L. et al.2021, Pharmaceutics 2021, 13, 1762). Furthermore, FGF-2 can be degraded at acidic pH, and is susceptible to degradation by proteases. Moreover, FGF-2 is prone to aggregation which can lead to loss of activity (Benington, L. et al. 2021, Pharmaceutics 2021, 13, 1762).

[0009] Growth factors used in skin care formulations can be derived from a variety of human sources, including adipose tissue, umbilical cords, and foreskin. They may also originate from engineered sources, such as like human stem cells and platelet-rich plasma. In addition, bioengineered non-human sources, such as bacteria, yeast, plants, and snail mucin, can be used to produce growth factors. Growth factor containing ingredients are typically incorporated in skin care formulations, as extracts, partially purified substances, or purified proteins. However, current sources of growth factors present several limitations. These include high production costs, inconsistent purity – particularly for partially purified or crude extracts – and inefficient manufacturing processes. For example, expressing human FGF-2 in Escherichia coli yields low quantities, due to complex processing steps like refolding, purification, and requirements to remove E. coli endotoxins (Sauer, D.G. et al., 2019, Protein Expression and Purification 153, 70-82).

[0010] These challenges underscore unmet needs in ingredient formulation, compounding, and delivery of growth factors, highlighting the demand for affordable, and reliably pure recombinant growth factors to enhance the effectiveness of topical formulations.

[0011] Accordingly, there remains a clear need in the art for novel topically applied formulations comprising growth factors, especially FGF-2, that are both effective in treating skin aging and wound healing, and for techniques and ingredients for preparing such formulations. SUMMARY OF THE DISCLOSURE

[0012] The following paragraphs are intended to introduce the reader to the more detailed description, not to define or limit the claimed subject matter of the present disclosure.

[0013] In one aspect, the present disclosure relates to dermal topical formulations.

[0014] In another aspect, the present disclosure relates to ingredients used in the preparation of dermal topical formulations, in particular, bioactive growth factors.

[0015] In another aspect, the present disclosure relates to further ingredients used in the preparation of dermal topical formulations, in particular oleaginous ingredients obtainable from plants and known as oleosomes.

[0016] Accordingly, in one aspect, the present disclosure provides, in at least one embodiment, in accordance with the teachings herein, a dermal topical formulation comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to stimulate dermal collagen production, together with a dermally acceptable diluent, excipient, or carrier, or mixture thereof.

[0017] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be fused to an oleosome associated polypeptide, the FGF-2 polypeptide and the oleosome associated polypeptide forming a single contiguous polypeptide.

[0018] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be fused to an oleosome associated polypeptide, wherein the FGF-2 polypeptide and the oleosome associated polypeptide are separated by a cleavable polypeptide linker, the oleosome associated polypeptide, the cleavable polypeptide linker, and the FGF-2 polypeptide forming a single contiguous polypeptide.

[0019] In an aspect, in at least one embodiment, the oleosome associated protein can be an oleosin, a caleosin, or a steroleosin.

[0020] In an aspect, in at least one embodiment, the FGF-2 polypeptide can constitute from about 0.00005% (w / w) up to about 0.5% (w / w) of the formulation.

[0021] In an aspect, in at least one embodiment, the FGF-2 polypeptide can constitute from about 0.00010% (w / w) up to about 0.0005% (w / w) of the formulation.

[0022] In an aspect, in at least one embodiment, the FGF-2 polypeptide can constitute from about 0.00015% (w / w) up to about 0.00025% (w / w) of the formulation.

[0023] In an aspect, in at least one embodiment, the formulation can contain from about 0.0001% (w / w) up to about 10% (w / w) oleosomes.

[0024] In an aspect, in at least one embodiment, the formulation can contain from about 0.005% (w / w) up to about 0.6% (w / w) oleosomes.

[0025] In an aspect, in at least one embodiment, the formulation can contain from about 0.01% (w / w) up to about 0.05% (w / w) oleosomes.

[0026] In an aspect, in at least one embodiment, the formulation can contain from about 90%% (w / w) up to about 99.9% (w / w) of the diluent, excipient, or carrier, or mixture thereof.

[0027] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be emulsified by a plant oleosome obtained from the group of plant species consisting of almond (Prunus dulcis), camelina (Camelina sativa), hemp (Cannabis sativa), linseed / flax (Linum usitatissimum), mustard (Brassica spp. and Sinapis alba), rapeseed (Brassica spp.), safflower (Carthamus tinctorius), and sunflower (Helianthus annuus).

[0028] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be emulsified by a plant oleosome obtained from a plant belonging to the plant genus Camelina.

[0029] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be emulsified by a plant oleosome obtained from a plant belonging to the plant species Camelina sativa.

[0030] In an aspect, in at least one embodiment, the dermal topical formulation can further comprise a preservative agent.

[0031] In an aspect, in at least one embodiment, the dermal topical formulation can further comprise at least one of an additional formulary ingredient selected from a moisturizer, an emollient, a surfactant, a viscosity modifying agent, or a chelating agent.

[0032] In an aspect, in at least one embodiment, the dermal topical formulation can be a cream, a crème, a gel, a foam, an ointment, a paste, a lotion, a serum, a tincture, a milk, a fluid, or a powder.

[0033] In an aspect, in at least one embodiment, the dermal topical formulation can be a formulation for the prevention, amelioration, or treatment of a skin condition,

[0034] In an aspect, in at least one embodiment, the skin condition can be a condition associated with skin aging.

[0035] In an aspect, in at least one embodiment, the skin condition associated with skin aging can be selected from skin aging spots, wrinkles,pigmentation, loss of skin tone, rough skin texture, uneven skin texture, broken capillaries, redness, weak skin barrier, and blotchiness.

[0036] In an aspect, in at least one embodiment, the skin condition can be a condition associated with skin injury.

[0037] In an aspect, in at least one embodiment, the dermal topical formulation can stimulate skin fibroblast cell proliferation to thereby effect healing of injured skin.

[0038] In an aspect, in at least one embodiment, the skin condition associated with skin injury can be an acute wound.

[0039] In an aspect, in at least one embodiment, the acute wound can be a superficial burn injury, a deep burn injury, skin trauma, or a surgical incision wound.

[0040] In an aspect, in at least one embodiment, the skin condition associated with skin injury can be alopecia, psoriasis, atopic dermatitis, acne, or a diabetic foot ulcer.

[0041] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a method of making a dermal topical formulation for the prevention, treatment, or amelioration of a skin condition in a subject, the method comprising: providing oleosome-emulsified FGF-2; providing a dermally acceptable diluent, excipient, or carrier, or mixture thereof; and contacting the oleosome-emulsified FGF-2 with the dermally acceptable diluent, excipient, or carrier to prepare a dermal topical formulation comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF- 2) polypeptide to stimulate dermal collagen production.

[0042] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be fused to an oleosome associated polypeptide, the FGF-2 polypeptide and the oleosome associated polypeptide forming a single contiguous polypeptide.

[0043] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be fused to an oleosome associated polypeptide, wherein the FGF-2 polypeptide and the oleosome associated polypeptide are separated by a cleavable polypeptide linker, the oleosome associated polypeptide, thecleavable polypeptide linker, and the FGF-2 polypeptide forming a single contiguous polypeptide.

[0044] In an aspect, in at least one embodiment, the oleosome associated protein can be an oleosin, a caleosin, or a steroleosin.

[0045] In an aspect, in at least one embodiment, the FGF-2 polypeptide can constitute from about 0.00005% (w / w) up to about 0.5% (w / w) of the formulation.

[0046] In an aspect, in at least one embodiment, the FGF-2 polypeptide can constitute from about 0.000.1% (w / w) up to about 0.0005% (w / w) of the formulation.

[0047] In an aspect, in at least one embodiment, the FGF-2 polypeptide can constitute from about 0.00015% (w / w) up to about 0.00025% (w / w) of the formulation.

[0048] In an aspect, in at least one embodiment, the formulation can contain from about 0.0001% (w / w) up to about 10% (w / w) oleosomes.

[0049] In an aspect, in at least one embodiment, the formulation can contain from about 0.005% (w / w) up to about 0.6% (w / w) oleosomes.

[0050] In an aspect, in at least one embodiment, the formulation can contain from about 0.01% (w / w) up to about 0.05% (w / w) oleosomes.

[0051] In an aspect, in at least one embodiment, the formulation can contain from about 90% (w / w) up to about 99.9% (w / w) of the diluent, excipient, or carrier, or mixture thereof.

[0052] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be emulsified by a plant oleosome obtained from the group of plant species consisting of almond (Prunus dulcis), camelina (Camelina sativa), hemp (Cannabis sativa), linseed / flax (Linum usitatissimum), mustard (Brassica spp. and Sinapis alba), rapeseed (Brassica spp., including, without limitation, the canola genotype), safflower (Carthamus tinctorius), and sunflower (Helianthus annuus).

[0053] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be emulsified by a plant oleosome obtained from a plant belonging to the plant genus Camelina.

[0054] In an aspect, in at least one embodiment, the FGF-2 polypeptide can be emulsified by a plant oleosome obtained from a plant belonging to the plant species Camelina sativa.

[0055] In an aspect, in at least one embodiment, the dermal topical formulation can further comprise a preservative agent.

[0056] In an aspect, in at least one embodiment, the dermal topical formulation can further comprise at least one of an additional formulary ingredient selected from a moisturizer, an emollient, a surfactant, a viscosity modifying agent, or a chelating agent.

[0057] In an aspect, in at least one embodiment, the dermal topical formulation can be a cream, a crème, a gel, a foam, an ointment, a paste, a lotion, a serum, a tincture, or a powder.

[0058] In at least one embodiment, in an aspect, the skin condition can be a condition associated with skin aging.

[0059] In an aspect, in at least one embodiment, the skin condition associated with skin aging can be selected from skin aging spots, wrinkles, pigmentation, loss of skin tone, rough skin texture, uneven skin texture, broken capillaries, redness, weak skin barrier, and blotchiness.

[0060] In an aspect, in at least one embodiment, the skin condition can be a condition associated with skin injury.

[0061] In an aspect, in at least one embodiment, the dermal topical formulation can stimulate skin fibroblast cell proliferation to thereby effect healing of injured skin.

[0062] In an aspect, in at least one embodiment, the skin condition associated with skin injury can be an acute wound.

[0063] In aspect, in at least one embodiment, the acute wound can be a superficial burn injury, a deep burn injury, skin trauma, or a surgical incision wound.

[0064] In an aspect, in at least one embodiment, the skin condition associated with skin injury can be alopecia, psoriasis, atopic dermatitis, acne, or a diabetic foot ulcer.

[0065] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a method of preventing, treating, or ameliorating a skin condition, the method comprising administeringto a subject in need thereof a formulation for dermal topical application comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to stimulate dermal collagen production, together with a dermally acceptable diluent, excipient, or carrier, or mixture thereof, wherein the formulation is administered in an effective amount to prevent, treat, or ameliorate the dermal condition in the subject.

[0066] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a use of a formulation for dermal topical application for the prevention, treatment, or amelioration of a skin condition in a subject in need thereof, the formulation comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to stimulate dermal collagen production, together with a dermally acceptable diluent, excipient, or carrier, or mixture thereof, and wherein the formulation is for use in an effective amount to prevent, treat or ameliorate the dermal condition in the subject.

[0067] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a formulation for dermal topical application for use in the prevention, treatment, or amelioration of a skin condition in a subject in need thereof, the formulation comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to stimulate dermal collagen production, together with a dermally acceptable diluent, excipient, or carrier, or mixture thereof, and wherein the formulation is for use in an effective amount to prevent, treat or ameliorate the dermal condition in the subject. In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a use of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to prepare a formulation for dermal topical application for the prevention, treatment, or amelioration of a skin condition in a subject in need thereof, the formulation comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to stimulate dermal collagen production, together with a dermally acceptable diluent, excipient, or carrier, or mixture thereof.

[0068] In an aspect, in at least one embodiment, the skin condition can be a condition associated with skin aging.

[0069] In an aspect, in at least one embodiment, the skin condition associated with skin aging can be selected from skin aging spots, wrinkles, pigmentation, loss of skin tone, rough skin texture, uneven skin texture, broken capillaries, redness, weak skin barrier, and blotchiness.

[0070] In an aspect, in at least one embodiment, the skin condition can be a condition associated with skin injury.

[0071] In an aspect, in at least one embodiment, skin condition associated with skin injury can be an acute wound.

[0072] In in aspect, in at least one embodiment, the acute wound can be a superficial burn injury, a deep burn injury, trauma, or a surgical incision wound.

[0073] In an aspect, in at least one embodiment, the skin condition associated with skin injury can be alopecia, psoriasis, atopic dermatitis, acne, or a diabetic foot ulcer.

[0074] In an aspect, in at least one embodiment, the subject can be a human subject.

[0075] Other features and advantages will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating preferred implementations of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The disclosure is in the hereinafter provided paragraphs described, by way of example, in relation to the attached figures. The figures provided herein are provided for a better understanding of the example embodiments and to show more clearly how the various embodiments may be carried into effect. The figures are not intended to limit the present disclosure.

[0077] FIG.1 depicts a bar graph representing certain results obtained in the performance of certain experiments, notably experiments to assay collagen Type 1 expression in tissue after dermal topical application of FGF-2 containing materials, notably unblended oleosome-emulsified FGF-2 at different concentrations (1.887 μg / ml; 0.472 μg / ml; 0.0944 μg / ml; 0.0472 μg / ml;and 0.00944 μg / ml) and blended (formulated) oleosome-emulsified FGF-2 (concentrations of 1 μg / ml and 0.1 μg / ml). The results for an untreated tissue control are also shown (bar on the far left).

[0078] FIG.2 depicts a bar graph representing certain results obtained in the performance of certain experiments, notably experiments to assay collagen Type 1 expression in tissue after dermal topical application of FGF-2 containing materials, notably unblended oleosome-emulsified FGF-2 at different concentrations (1.887 μg / ml; 0.472 μg / ml; 0.0944 μg / ml; 0.0472 μg / ml; and 0.00944 μg / ml) and blended (formulated) oleosome-emulsified FGF-2 (concentrations of 1 μg / ml and 0.1 μg / ml), following 48 hours (lighter shaded bars) and 96 hours (darker shaded bars) of application. The results for an untreated tissue control are also shown (2 bars on the far left).

[0079] FIGS.3A, 3B, and 3C depict certain graphs representing certain results obtained in the performance of certain experiments, notably experiments to assay 3T3 cell proliferation, notably a graph (FIG.3A) assaying absorption at 450 nm (vertical axis) using various concentrations (horizontal axis) of FGF- 2 containing materials, notably purified FGF-2 (designated “rhFGF2”) immediately following constitution; oleosome-emulsified FGF-2 ( designated “oleo-CLS-rhFGF2”); oleo-CLS-FGF2 following 5 weeks of storage at 4oC; and rhFGF2 following 5 weeks of storage at 4oC; a bar graph (FIG.3B) assaying proliferation of 3T3 cells of FGF-2 containing materials, notably rhFGF2 (lighter shaded bars) and oleo-CLS-rhFGF2 purified oleosomes (darker shaded bars) following incubation at 37oC for 6 hours, 12 hours, 24 hours, 48 hours and 72 hours; and a bar graph (FIG.3C) assaying proliferation of 3T3 cells of FGF-2 containing materials, notably rhFGF2 (set of bars on the far left) and oleo-CLS- rhFGF2 purified oleosomes (set of bars on the middle left), wild type oleosome control (set of bars on the middle right), and NBCS control (set of bars on the far right) following incubation at 37oC for 0 hours (lightest shaded bars), 24 hours (intermediate lighter grey shaded bars), 48 hours (intermediate darker grey shaded bars) and 72 hours (darkest shaded bars).

[0080] FIG.4 depicts a Coomassie blue stained gel prepared in a gel electrophoresis experiment to visualize proteins separated by mass present in an oleosome sample (left) and a graph (right) representing certain resultsobtained in the performance of certain experiments, notably experiments to assay for the presence of oleosin FGF-2 fusion protein in an oleosome sample, and to assay the stability of oleosome-emulsified FGF-2 (designated “FGF-2 fusion protein”) relative to non-emulsified purified FGF-2 (designated “naked FGF-2”) over time up to 12 months (horizontal axis).

[0081] The figures together with the following detailed description make apparent to those skilled in the art how the disclosure may be implemented in practice. DETAILED DESCRIPTION

[0082] Various compositions, systems or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below. The claimed subject matter is not limited to compositions, processes or systems having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system, or process described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in a composition, system or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.

[0083] As used herein and in the claims, the singular forms, such “a”, “an” and “the” include the plural reference and vice versa unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, “comprise,” “comprises” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

[0084] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and sub-combinations of ranges and specific embodimentstherein are intended to be included. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range, as will be readily recognized by context. Furthermore, any range of values described herein is intended to specifically include the limiting values of the range, and any intermediate value or sub-range within the given range, and all such intermediate values and sub- ranges are individually and specifically disclosed (e.g., a range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). Similarly, other terms of degree such as "substantially" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.

[0085] Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0086] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Terms and definitions

[0087] The term “oleosomes” as used herein, refers to natural lipid containing compartments situated within and obtainable from plant cells, comprised generally of a core of triglycerides surrounded by a monolayer of phospholipids embedded with oleosome associated polypeptides, typicallyoleosins (including, for example, a polypeptide set forth herein having SEQ.ID NO: 6), caleosins (including, for example, a polypeptide set forth herein having SEQ.ID NO: 14), and steroleosins (including, for example, a polypeptide set forth herein having SEQ.ID NO: 20). Oleosomes (sometimes called “oil bodies”, “oilbodies”, or “oil-bodies”) of different plant species have similar compositions and structures. The properties of the mixed phospholipid-protein layer at the surface of oleosomes have been found to make them particularly stable, for example, against coalescence and thermal processing. Oleosomes serve as lipid storage sites and as a source of energy during plant seed germination. They can be recovered from the cells of reproductive organs of different plants, including, without limitation, from oleaginous seeds, nuts and fruits, using aqueous processing techniques to provide oleosome compositions.

[0088] The term “oleosome associated polypeptide”, as used herein, refers to any polypeptide which forms part of an oleosome, as can be determined by assaying for the presence thereof in an oleosome fraction recovered from plant parts, and separated from other plant fractions, for example, by Enzyme-Linked-Immunosorbent Assay (ELISA) or immunoblotting. Oleosome associated polypeptides are generally embedded within the phospholipid membrane of the oleosome, and include oleosins (including, for example, a polypeptide set forth herein having SEQ.ID NO: 6), caleosins (including, for example, a polypeptide set forth herein having SEQ.ID NO: 14), and steroleosins (including, for example, a polypeptide set forth herein having SEQ.ID NO: 20), and in each case further include any polypeptides (i) that are substantially identical to an oleosome associated polypeptide, including a polypeptide substantially identical to a polypeptide having SEQ. ID NO: 6; SEQ. ID NO: 8; SEQ. ID NO: 10; SEQ. ID NO: 12; SEQ. ID NO: 14; SEQ. ID NO: 16; SEQ. ID NO: 18; and SEQ. ID NO: 20; and (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding an oleosome associated polypeptide set forth herein as SEQ. ID NO: 6; SEQ. ID NO: 8; SEQ. ID NO: 10; SEQ. ID NO: 12; SEQ. ID NO: 14; SEQ. ID NO: 16; SEQ. ID NO: 18; and SEQ. ID NO: 20, but for the use of synonymous codons, including the nucleic acid sequences set forth as SEQ. ID NO: 5; SEQ. ID NO: 7; SEQ. ID NO: 9; SEQ. ID NO: 11; SEQ. ID NO: 13; SEQ. ID NO: 15; SEQ. ID NO: 17; and SEQ. ID NO: 19.

[0089] The terms “fibroblast growth factor 2” or “FGF-2” or “FGF2” or “FGF-2 polypeptide”, as may be used herein interchangeably, refer to any and all proteins that have the activity of fibroblast growth factor 2, including, but not limited to proteins comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequence of the FGF-2 polypeptide set forth herein as SEQ.ID NO: 2, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding the FGF-2 polypeptide set forth herein as SEQ.ID NO: 2, but for the use of synonymous codons, including the nucleic acid sequence set forth as SEQ.ID NO: 1. The term further includes mature FGF-2 polypeptides, including SEQ.ID NO: 2, for example, and FGF-2 polypeptides including a pro-sequence, including SEQ.ID NO: 4, for example.

[0090] By the term “substantially identical” it is meant that two amino acid sequences preferably are at least 70% identical, at least 75% identical, or at least 80% identical, and more preferably are at least 85% or 90% identical, and most preferably at least 95% identical, for example 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical. In order to determine the percentage of identity between two amino acid sequences the amino acid sequences of such two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol., 1990:215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J. D., Higgines, D. G. and Gibson T. J / , 1994, NucleicAcid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of the two sequences is involved in the alignment.

[0091] By the term “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600 / l), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5× sodium chloride / sodium citrate (SSC) / 5×Denhardt's solution / 1.0% SDS at Tm (based on the above equation) −5° C, followed by a wash of 0.2×SSC / 0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3×SSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts, and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol.3.

[0092] The term “Camelina”, as used herein, refers to a plant belonging to the taxonomically classified genus Camelina and includes, without limitation, the taxonomically classified species Camelina sativa, also known as camelina, gold-of-pleasure, false flax, wild flax, linseed dodder, German sesame, or Siberian oilseed, and Camelina alyssum, Camelina microcarpa, and Camelina rumelica, and further includes all plant cultivars, varieties, and genotypes belonging to the aforementioned.

[0093] The term “dermal topical formulation”, as used herein, refers to a preparation in a form which allows an active ingredient, including, for example, FGF-2, contained therein, to provide effective treatment, and which does not contain any other ingredients which cause excessive toxicity, an allergic response, irritation, or other adverse response commensurate with a reasonable risk / benefit ratio. The dermal topical formulation may contain other ingredients such as excipients, carriers, diluents, or formulary agents.

[0094] The term “effective amount”, as used herein, refers to an amount of an active agent or formulation, sufficient to induce a desired biological or therapeutic effect, including a prophylactic effect, and further including an effect resulting in the improvement of a skin condition, including a skin condition associated with aging or injury. Such effect can include an effect with respect to the signs, symptoms or causes of a disorder, or condition or any other desired alteration of a biological system. The effective amount can vary depending, for example, on the health condition, injury stage, disorder stage, or disease stage, weight, or sex of a subject being treated, timing of the administration, manner of the administration, age of the subject, and the like, all of which can be determined by those of skill in the art.

[0095] The terms “treating” and “treatment”, and the like, as used herein, are intended to mean obtaining a desirable physiological, pharmacological, or biological effect, and includes prophylactic and therapeutic treatment. The effect may result in the inhibition, attenuation, amelioration, or reversal of a sign, symptom or cause of a disorder, or condition, attributable to the disorder, or condition, which includes, without limitation, skin conditions associated with aging or injury. Clinical evidence or other efficacy evidence of the prevention or treatment may vary with the disorder, or condition, the subject, and the selected treatment.

[0096] The term “dermatologically acceptable”, as used herein, refers to materials, including excipients, carriers, diluents, or other formulary ingredients, that are compatible with other materials in a dermal topically applied formulation and within the scope of reasonable medical judgement suitable for use in contact with a subject without excessive toxicity, allergic response, irritation, or other adverse response commensurate with a reasonable risk / benefit ratio.

[0097] The term “subject”, as used herein, refers to all members of the kingdom Animalia, and includes humans.

[0098] The terms “substantially pure” and “isolated”, as may be used interchangeably herein describe a compound, e.g., an oleosome or growth factor, which has been separated from components that naturally or synthetically accompany it. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., by chromatography, gel electrophoresis or HPLC analysis. General implementation

[0099] As hereinbefore mentioned, the present disclosure relates to dermal topical formulations. Furthermore, the present disclosure relates to ingredients used in the preparation of dermal topically applied formulations, notably FGF-2 and plant oleosomes. In broad terms, the present disclosure provides formulations for dermal topical application comprising an oleosome- emulsified FGF-2 for the prevention, treatment, or amelioration of skin conditions, and methods of making and using the same.

[0100] The formulations of the present disclosure are particularly useful since they provide FGF-2 polypeptide in a stable form, i.e., a form where FGF- 2 retains its biological activity. The same is of significance since FGF-2 is known to be a protein that is highly susceptible to thermal, acidic, and proteolytic degradation. Thus, for example, at 4 °C solutions of FGF-2 may stable for only a few hours (Benington, L. et al. 2021, Pharmaceutics 2021, 13, 1762). By contrast, and surprisingly, within the formulations of the present invention, FGF- 2 retains its biological activity and hence the formulations of the presentdisclosure can be stored for periods of time (e.g., at least up to 12 months) and under conditions (e.g., at room temperature) that are required for commercial development and sale of products containing FGF-2.

[0101] Furthermore, FGF-2’s biological activity is retained upon dermal topical application of the formulations of the present disclosure, and, surprisingly, FGF-2, when formulated as described herein, can traverse the skin barrier, notably the stratum corneum. Moreover, when formulated as herein described, FGF-2 can penetrate to the deeper dermal layers of the skin, and can surprisingly retain its biological activity, and act to stimulate collagen production in the dermal skin layers.

[0102] Furthermore, FGF-2’s biological activity is retained upon dermal application to injured skin so that FGF-2 can stimulate skin fibroblast cell proliferation to thereby effect wound healing.

[0103] Furthermore, the key components of the formulations of the present disclosure, notably oleosome-emulsified FGF-2, can readily be produced in large quantities, and sourced from natural materials. The use of natural materials is highly valued in the development of products for dermal topical application.

[0104] In view of the foregoing, the formulations of the present disclosure are particularly useful for the treatment of skin conditions, including conditions associated with skin aging, and conditions associated with skin injury.

[0105] In what follows selected embodiments are described.

[0106] Accordingly, in one aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a dermal topical formulation comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to stimulate dermal collagen production, together with a dermally acceptable diluent, excipient, or carrier.

[0107] Thus, according to an aspect of the present disclosure, dermal topical formulations comprising an oleosome-emulsified FGF-2 polypeptide are provided. The formulations of the present disclosure are suitable for administration to a subject in need thereof to treat skin conditions. Thus, it will be clear that the formulations of the present disclosure are prepared to contain FGF-2 polypeptide and oleosomes.

[0108] Next, initially suitable example compositions comprising FGF-2 polypeptide and suitable example compositions comprising oleosomes and techniques for obtaining the same will be described. Thereafter example techniques to obtain oleosome-emulsified FGF-2 compositions, and dermal topical formulations containing the same will be described, as well as example methods for administering the same to subjects in need thereof to treat skin conditions. FGF-2 polypeptide

[0109] In accordance with one aspect, the formulations of the present disclosure are prepared to comprise FGF-2 polypeptide. By way of brief background, fibroblast growth factor 2 or FGF-2 is a signaling protein encoded by the FGF-2 gene (see: SEQ.ID NO: 1). In vivo FGF2 is involved in various biological processes in various cell and organ systems, including in cell proliferation, migration, and survival, and stimulation of dermal gene expression, for example, collagen expression. FGF-2 has also been implicated in angiogenesis, i.e., the formation of new blood vessels, and cancer (see, for example, Kottakis, F. et al., 2011, Molecular Cell 43, 285-298.

[0110] Initially, considering FGF-2 polypeptide, preparations containing an FGF-2 polypeptide can be prepared biosynthetically using a host cell system. In this respect, an isolated nucleic acid encoding an amino acid sequence corresponding with an FGF-2 polypeptide can be introduced in host cells and expressed therein. Nucleic acid sequences encoding mature and pro-sequence containing FGF polypeptides may be used, SEQ.ID. NO: 1 and SEQ.ID NO: 3, respectively, for example.

[0111] According to an aspect, in an example embodiment, a nucleic acid sequence encoding an FGF-2 polypeptide may be selected, wherein such nucleic acid includes SEQ.ID NO: 1 set forth herein. A selected example FGF- 2 polypeptide includes SEQ.ID NO: 2, which is a polypeptide encoded by SEQ.ID NO: 1. A further selected example FGF-2 polypeptide includes SEQ.ID NO: 4, which is a polypeptide encoded by SEQ.ID NO: 3. It is noted that FGF- 2 polypeptides including a pro-sequence, such as SEQ.ID NO: 4, generally require maturation, i.e., separation of the mature polypeptide from the pro- sequence. Further example FGF-2 polypeptides that may be used inaccordance herewith are FGF-2 polypeptides which are substantially identical to SEQ.ID NO: 2 or SEQ.ID NO: 4, for example, polypeptides that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ.ID NO: 2 or SEQ.ID NO: 4. A nucleic acid sequence encoding FGF-2 initially may be isolated from a natural source, for example, from a human cDNA library. Alternatively, such a nucleic acid sequence may be chemically synthesized using techniques that are well known to those of skill in the art, for example, solid-phase (controlled pore glasses, for example) oligonucleotide synthesis, using, for example, an automated DNA synthesizer based on phosphoramidite chemistry.

[0112] As is further known to those of skill in the art, expression of nucleic acids in a host cell, to thereby biosynthetically produce a protein, can be achieved by providing one or more nucleic acids capable of controlling expression in a host cell, and operably linking the one or more nucleic acids capable of controlling expression in a host cell to the nucleic acid one wishes to express. Such operable linking of a nucleic acid controlling expression generally involves linking in the 5’ to 3’ direction of expression the nucleic acid capable of controlling expression in a host cell to the nucleic acid one wishes to express. Thus, within the context of the instant disclosure, a nucleic acid encoding an FGF-2 polypeptide, including, for example SEQ.ID NO: 1 can be linked to a nucleic acid controlling expression in a host cell. Suitable nucleic acid sequences capable of controlling expression in host cells that may be used herein include any transcriptional promoter capable of controlling expression of polypeptides in host cells. Generally, promoters obtained from bacterial cells are used when a bacterial host cell is selected, while a yeast promoter will be used when a yeast host cell is selected, an animal cell promoter will be used when an animal cell is selected, and so on. The obtained nucleic acid comprising a promoter and the nucleic acid expressing an FGF-2 polypeptide is generally a chimeric nucleic acid. Further nucleic acid elements capable elements of controlling expression in a host cell include transcriptional terminators, enhancers and the like, all of which may be included in the chimeric nucleic acid sequences of the present disclosure.

[0113] In accordance with an aspect of the present disclosure, the chimeric nucleic acid sequences including a nucleic acid sequence expressingan FGF-2 polypeptide can be integrated into a recombinant expression vector which ensures good expression in the host cell, wherein the recombinant expression vector is suitable for expression in a host cell. The term “suitable for expression in a host cell” means that the recombinant expression vector comprises the chimeric nucleic acid linked to genetic elements required to achieve expression in a cell. As noted, such genetic elements can include transcriptional promoters, terminators and enhancers, and the like. Further genetic elements that may be included in the expression vector are one or more nucleic acid sequences encoding marker genes, and one or more origins of replication. In some embodiments, the expression vector can freely replicate in the host cell. In other embodiments, the chimeric nucleic acid can be integrated into the host cell’s genomic DNA. In some embodiments, the expression vector further can comprise genetic elements required for the integration of the vector or a portion thereof in the host cell's genome.

[0114] Marker genes that may be used in accordance with the present disclosure include all genes that allow the distinction of transformed cells from non-transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin, chloramphenicol, methotrexate, or ampicillin. In other instances, a marker gene may be a gene which allows a cell to produce an essential nutrients, for example amino acids.

[0115] Turning now to the host cell, it is noted, initially, that any host cell which upon cultivation expresses the nucleic acid sequence encoding an FGF- 2 polypeptide can be selected and used. Suitable host cells in this respect include, for example, microbial cells, such as bacterial cells, yeast cells, for example, and algal cells or animal cells. A variety of techniques and methodologies to manipulate host cells to introduce nucleic acid sequences in cells and attain expression exists and are well known to the skilled artisan. These methods include, for example, cation based methods, for example, lithium ion or calcium ion based methods, electroporation, biolistics, and glass beads based methods. As will be known to those of skill in the art, depending on the host cell selected, the methodology to introduce nucleic acid material in the host cell may vary, and, furthermore, methodologies may be optimized for uptake of nucleic acid material by the host cell, for example, by comparinguptake of nucleic acid material using different conditions. Detailed guidance can be found, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. It is noted that the chimeric nucleic acid is a non-naturally occurring chimeric nucleic acid sequence and can be said to be heterologous to the host cell.

[0116] One example host cell that conveniently may be used is Escherichia coli. The preparation of the E. coli vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing, the polymerase chain reaction (PCR) and other methodologies. A wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector. Among the vectors with a replication system functional in E. coli, are vectors such as pBR322, the pUC series of vectors, the M13 mp series of vectors, pBluescript etc. Suitable promoter sequences for use in E. coli include, for example, the T7 promoter, the T5 promoter, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan / lactose (tac) promoter, lipoprotein (Ipp) promoter, and λ phage PL promoter. Typically, cloning vectors contain a marker, for example, an antibiotic resistance marker, such as ampicillin or kanamycin resistance marker, allowing selection of transformed cells. Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in E. coli by preparing competent cells, electroporation or using other well-known methodologies to a person of skill in the art. E. coli may be grown in an appropriate medium, such as Luria-Broth medium and harvested. Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells.

[0117] Another example host cell that may be conveniently used is a yeast cell. Example yeast host cells that can be used are yeast cells belonging to the genus Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansenula, and Yarrowia. In specific example embodiments, the yeast cell can be a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, or Pichia pastoris cell.

[0118] A number of vectors exist for the expression of recombinant proteins in yeast host cells. Examples of vectors that may be used in yeast host cells include, for example, Yip type vectors, YEp type vectors, YRp type vectors,YCp type vectors, pGPD-2, pAO815, pGAPZ, pGAPZα, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, pPICZ, pPICZα, pPIC3K, pHWO10, pPUZZLE and 2 µm plasmids. Such vectors are known to the art and are, for example, described in Cregg et al., Mol Biotechnol. (2000) 16(1): 23-52. Suitable promoter sequences for use in yeast host cells are also known and described, for example, in Mattanovich et al., Methods Mol. Biol., 2012, 824:329-58, and in Romanos et al., 1992, Yeast 8: 423-488. Examples of suitable promoters for use in yeast host cells include promoters of glycolytic enzymes, like triosephosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase (GAL), P. pastoris glucose-6- phosphate isomerase promoter (PPGI), the 3-phosphoglycerate kinase promoter (PPGK), the glycerol aldehyde phosphate dehydrogenase promoter (PGAP), translation elongation factor promoter (PTEF), S. cerevisiae enolase (ENO-1), S. cerevisiae galactokinase (GAL1), S. cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), S. cerevisiae triose phosphate isomerase (TPI), S. cerevisiae metallothionein (CUP1), and S. cerevisiae 3-phosphoglycerate kinase (PGK), and the maltase gene promoter (MAL). Marker genes suitable for use in yeast host cells are also known to the art. Thus, antibiotic resistance markers, such as ampicillin resistance markers, can be used in yeast, as well as marker genes providing genetic functions for essential nutrients, for example, leucine (LEU2), tryptophan (TRP1 and TRP2), uracil (URA3, URA5, URA6), histidine (HIS3), and the like. Methods for introducing vectors into yeast host cells can, for example, be found in S. Kawai et al., 2010, Bioeng. Bugs 1(6): 395-403.

[0119] A further example of host cells that may be used in accordance herewith are animal host cells. These include, for example, mammalian cells, such as Chinese Hamster Ovary cells (CHO) cells, or lymphoid cells (e.g., Υ0, NS0, or Sp20 cells), which are able to grow and survive when placed in either monolayer culture or suspension culture in medium containing appropriate nutrients and / or growth factors. Examples of expression vectors suitable for expression in animal cells include, but are not limited to, BPV-1, pHyg, pRSV, pIRES (Clontech), and pSG5 vectors (Stratagene). Selectable markers can used in to confer resistance to the cells harboring the vector to allow theirselection in appropriate selection medium. A number of selection systems can be used, including but not limited to, the Herpes Simplex Virus thymidine kinase (HSV TK), (Wigler et al., 1977, Cell, 11:223), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), (Szybalska and Szybalski, 1992, Proc. Natl. Acad. Sci. USA, 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell, 22:817) genes. Further vectors, media and growth conditions for animal cells can be found in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0120] Yet other example host cells that may be used in accordance herewith are plant cells. Methods for introducing nucleic acids in plant cells are known to those of skill in the art. Agrobacterium mediated plant cell transformation methods are described, for example, by Gelvin S. in Microbiol. Mol. Biol. Rev., 2003, 67(1): 16-37, and physical transformation based methods for plant cells are described by Rivera A.L. et al., 2012, Phys. Life Rev.9(3): 308-345. Plant selectable marker genes are known to those of skill in the art and include antibiotic resistance genes, for example kanamycin resistance genes, and herbicide resistance genes, such as the bar and pat genes (Wohlleben et al., 1988, Gene 70:25-37). Screenable markers that may be employed to identify plant transformants through visual inspection include β- glucuronidase (GUS) (U.S. Pat. Nos. 5,268,463 and 5,599,670) and green fluorescent protein (GFP) (Niedz et al., 1995, Plant Cell Rep., 14: 403). Plant promoters are also known to those in the art and include, for example, constitutive promoters, such as the 35S cauliflower mosaic virus (CaMV) promoter (Rothstein et al., 1987, Gene 53: 153-161), the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171; U.S. Pat. No.6,429,357), a ubiquitin promoter, such as the corn ubiquitin promoter (U.S. Pat. Nos 5,879,903 and 5,273,894), and the parsley ubiquitin promoter (Kawalleck, P. et al., 1993, Plant Mol. Biol. 21:673-684), and organ specific promoters, such as seed specific promoters, for example, a phaseolin promoter (Sengupta-Gopalan et al., 1985, Proc. Natl. Acad. Sci. USA 82: 3320-3324), or an oleosin promoter (U.S. Pat. No.5,792,922).

[0121] Furthermore, in one preferred embodiment, a nucleic acid sequence encoding an FGF-2 polypeptide, may be operably fused to a nucleic acid sequence encoding an oleosome associated polypeptide, for example, anucleic acid sequence encoding an oleosin (for example, an Arabidopsis oleosin, for example, SEQ.ID NO: 5; or a Camelina sativa oleosin, SEQ.ID NO: 7; SEQ.ID NO: 9; or SEQ.ID NO: 11, for example), a caleosin (for example, a Camelina sativa oleosin, SEQ.ID NO: 13; SEQ.ID NO: 15; or SEQ.ID NO: 17, for example), or steroleosin (for example, SEQ.ID NO: 19), and operably linked, for example, to a promoter, for example, a seed specific promoter, and used to transform a plant cell, for example, a Camelina sativa plant cell, a Prunus dulcis plant cell, a Cannabis sativa plant cell, a Linum usitatissimum plant cell, a Brassica spp. Plant cell, a Sinapis alba plant cell, a Carthamus tinctorius plant cell, or a Helianthus annuus plant cell. An example nucleic acid sequence encoding an oleosin operably fused to a nucleic acid sequence encoding an FGF-2 polypeptide, and the amino acid sequence of the pertinent fusion polypeptide are set forth in SEQ.ID NO: 23 and SEQ.ID NO: 24, respectively. Techniques for fusing polypeptides to oleosome associated polypeptides (C- terminally or N-terminally), and expressing the same in plant cells are further described, for example, in U.S. No. Patent 5,650,554 and by Van Rooijen, G. et al., 1995, Nature Biotechnology 13, 72 - 77. In some embodiments, the nucleic acid sequence encoding the oleosome associated polypeptide-FGF-2 fusion polypeptide may contain a nucleic acid sequence encoding a cleavable polypeptide linker separating the oleosome associated polypeptide and the FGF-2 polypeptide, for example, a chymosin, a trypsin, trypsinogen, or subtilisin cleavable polypeptide linker sequence, or a polypeptide sequence that is cleavable by a skin protease, such as kallikrein 5, kallikrein 7, cathepsin C, cathepsin D, cathepsin L or cathepsin V), or a polypeptide sequence that is acid cleavable. The amino acid sequence of an example chymosin linker and an example nucleic acid sequence encoding the same that may be used in accordance herewith include SEQ.ID NO: 26, and SEQ.ID NO: 25, respectively. Furthermore, an example nucleic acid sequence encoding an oleosin polypeptide operably fused to a nucleic acid sequence encoding an FGF-2 polypeptide wherein the fusion polypeptide comprises a chymosin linker sequence separating the oleosin polypeptide and FGF-2 polypeptide, and the amino acid sequence of the pertinent fusion polypeptide are set forth in SEQ.ID NO: 21 and SEQ.ID NO: 22, respectively. Expression in a host cell of nucleic acid sequence encoding an oleosome associated protein fused to a nucleic acidsequence encoding an FGF-2 polypeptide, results in the biosynthesis of a single contiguous oleosome associated polypeptide-FGF-2 polypeptide. The single contiguous oleosome associated polypeptide-FGF-2 polypeptide optionally comprises a cleavable polypeptide linker linking the oleosome associated polypeptide to the FGF-2 polypeptide.

[0122] Further, guidance with respect to the preparation of expression vectors and introduction thereof into host cells, may be found in, for example: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0123] Thus, to briefly recap, a host cell comprising a chimeric nucleic acid comprising (i) a nucleic acid sequence encoding an FGF-2 polypeptide, including, SEQ.ID NO: 1; and (ii) a nucleic acid sequence capable of controlling expression of the nucleic acid sequence encoding an FGF-2 polypeptide thereof in a host cell can be prepared in accordance with the present disclosure.

[0124] In accordance herewith, host cells are grown to multiply and to express a chimeric nucleic acid, including for example, in the case of plant cells to form a whole organism. Expression of the chimeric nucleic acid results in the biosynthetic production in the host cell of an FGF-2 polypeptide. Growth media and growth conditions can vary depending on the host cell that is selected, as will be readily appreciated to those of ordinary skill in the art. Growth media typically contain a carbon source, one or several nitrogen sources, essential salts including salts of potassium, sodium, magnesium, phosphate and sulphate, trace metals, water soluble vitamins, and process aids including but not limited to antifoam agents, protease inhibitors, stabilizers, ligands and inducers. Typical carbon sources are e.g., mono- or disaccharides. Typical nitrogen sources are, e.g., ammonia, urea, amino acids, yeast extract, corn steep liquor and fully or partially hydrolyzed proteins. Typical trace metals are e.g., Fe, Zn, Mn, Cu, Mo and H3BO3. Typical water soluble vitamins are e.g., biotin, pantothenate, niacin, thiamine, p-aminobenzoic acid, choline, pyridoxine, folic acid, riboflavin and ascorbic acid. Further, specific example media include liquid culture media for the growth of yeast cells and bacterial cells including, Luria-Bertani (LB) broth for bacterial cell cultivation, and yeast extract peptone dextrose (YEPD or YPD), for yeast cell cultivation, and CD-CHO medium, or Ham’s F10 medium for growing CHO cells. Further media and growth conditionscan be found in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0125] Upon production by the host cells of an FGF-2 polypeptide, the FGF-2 may be recovered from the host cells, and separated from other constituents, such as cellular debris, or media constituents, for example. Separation techniques will be known to those of skill in the art and include a variety of different protein purification techniques including, e.g., ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography, gel filtration, etc. It is noted that in view of the general lability of FGF-2 protein, in particular, when in solubilized form, purification conditions must be carefully controlled. In solubilized form FGF-2 is especially vulnerable to precipitation, and aggregation and loss of biological function. Thus, for example, during purification exposure to acidic conditions and proteolytic enzymes must be avoided, and purification steps are generally preferably conducted at lower temperatures, for example, at around 4oC. In addition, the obtained FGF-2 protein preparations are generally preferably stored at temperatures of -20oC or lower e.g., -70oC to -80oC, and preferably desiccated and / or in the presence of crypoprotectants (e.g., mannitol, glucose or polyethylene glycol), although in lyophilized form, FGF-2 proteins may be stored for a few days at 4oC. Further general guidance with respect to protein purification may for example be found in: Cutler, P. Protein Purification Protocols, Humana Press, 2004, Second Ed. Thus, substantially pure preparations of FGF-2 polypeptides may be obtained. The recovered FGF-2 polypeptide may be obtained in a more or less pure or substantially pure form, for example, a preparation of FGF-2 polypeptides of at least about 60% (w / v), about 70% (w / v), about 80% (w / v), about 90% (w / v), about 95% (w / v), or about 99% (w / v) purity may be obtained.

[0126] It is noted that the cells in some embodiments may secrete a portion of the produced FGF-2 polypeptide in the cell growth medium, thus a portion of the produced FGF-2 polypeptide may be recovered from the cells and a further portion of the FGF-2 polypeptide may be recovered from the growth medium.

[0127] It is further noted that when microbial expression systems are used, to express oleosome associated FGF-2 fusion polypeptides, the fusionpolypeptides may be expressed in inclusion bodies that can be recovered and emulsified with neutral and / or non-neutral lipids to generate synthetic oleosomes or oleosome like structures containing the fusion polypeptide embedded therein. Similarly fusion proteins may associate with lipids in other transgenic expression systems, such as yeast, fungus, mammalian or non-plant cells engineered to express the fusion protein, and oleosomes or oleosome-like structures containing the fusion polypeptide embedded therein may be recovered and purified. Oleosomes

[0128] Next, considering oleosomes, in accordance with one aspect hereof, the dermal topical formulations of the present disclosure comprise oleosomes. Oleosome preparations can be prepared from natural sources containing oleosomes. In some embodiments, oleosomes may be prepared from plants, plant materials, or plant parts including, without limitation, pollen, spores, seed, fruit, nuts and vegetative plant organs, in which oleosomes are present. Preferably, oleosomes are prepared from plant seeds, nuts or fruits. Suitable plants and plant seeds, nuts and fruits in accordance herewith are plants and plant seeds, nuts and / or fruits obtainable or obtained from the group of plant species consisting of: almond (Prunus dulcis), anise (Pimpinella anisum), avocado (Persea spp.), beach nut (Fagus sylvatica), borage (Boragio officinalis), Brazil nut (Bertholletia excelsa), camelina (Camelina sativa), candle nut (Aleuritis tiglium), carapa (Carapa guineensis), cashew nut (Ancardium occidentale), castor (Ricinus communis), coconut (Cocus nucifera), coriander (Coriandrum sativum), cottonseed (Gossypium spp.), crambe (Crambe abyssinica), Crepis alpine, croton (Croton tiglium), cucumber (Cucumis sativus), Cuphea spp., dill (Anethum gravealis), Euphorbia lagascae, evening primrose (Oenothera biennis), Dimorphoteca pluvialis, false flax (Camolina sativa), fennel (Foeniculum vulgaris), groundnut (Arachis hypogaea), hazelnut (coryllus avellana), hemp (Cannabis sativa), honesty plant (Lunnaria annua), jojoba (Simmondsia chinensis), kapok fruit (Ceiba pentandra), kukui nut (Aleuritis moluccana), Lesquerella spp., linseed / flax (Linum usitatissimum) including the solin genotype, lupin (Lupinus spp.), macademia nut (Macademia spp.), maize (Zea mays), meadow foam (Limnanthes alba), mustard (Brassica spp. andSinapis alba) including their canola genotypes, olive (Olea spp.), oil palm (Elaeis guineeis), oiticia (Licania rigida), paw paw (Assimina triloba), pecan (Juglandaceae spp.), perilla (Perilla frutescens), physic nut (Gatropha curcas), pilinut (Canarium ovatum), pine nut (pine spp.), pistachio (Pistachio vera), pongam (Bongamin glabra), poppy seed (Papaver somniferum), pumpkin (Cucurbita pepo), rapeseed (Brassica spp., including, without limitation, the canola genotype), safflower (Carthamus tinctorius), sesame seed (Sesamum indicum), soybean (Glycine max), squash (Cucurbita maxima), sal tree (Shorea rubusha), Stokes aster (Stokesia laevis), sunflower (Helianthus annuus), tukuma (Astocarya spp.); tung nut (Aleuritis cordata); vernonia (Vernonia galamensis); including any varieties, cultivars, genotypes or hybrids of any of the foregoing, and mixtures thereof.

[0129] In some embodiments, plant oleosomes may be prepared from the group of plant species consisting of almond (Prunus dulcis), camelina (Camelina sativa), hemp (Cannabis sativa), linseed / flax (Linum usitatissimum), mustard (Brassica spp. and Sinapis alba), rapeseed (Brassica spp.), safflower (Carthamus tinctorius), and sunflower (Helianthus annuus).

[0130] In some embodiments, plant oleosomes may be prepared from a plant belonging to the plant genus Camelina, notably, preferably from the seeds thereof.

[0131] In some embodiments, plant oleosomes may be prepared from a plant belonging to the plant species Camelina sativa, notably, preferably from the seeds thereof.

[0132] In some embodiments, plants or plant materials, such as seeds, nuts or fruits, may be obtained and used as a source material whence oleosomes are extracted. A wide variety of extraction processes and techniques may be used, provided however that, such extraction processes comprise conditions sufficiently gentle and mild to not, or not substantially, destroy the oleosome structures. Thus, in general, process techniques involving organic extractants, such as hexane, and high temperatures are less suitable. Suitable non-destructive extraction techniques include processes comprising (i) a comminution step, using for example stirring, milling or grinding processes and equipment, to disrupt plant tissues under conditions that do not substantially disrupt the integrity of the oleosome, and (ii) one or more aqueous extractionsteps, comprising mixing of the comminuted plant material with, for example, water or an aqueous buffer, and the separation of the oleosome fraction from insoluble plant materials, for example seed hulls and cell wall materials, and plant material soluble in the aqueous phase, for example water soluble plant proteins, simple sugars and water-soluble polysaccharides, using for example centrifugation equipment. The low density fraction thus obtained from the separation process comprises an oleosome suspension, which may be a substantially pure oleosome suspension, i.e., an oleosome suspension substantially free of other plant constituents, for example, an aqueous oleosome suspension comprising 10% (w / w) or less, 5% (w / w) or less, 4%, (w / w) or less, 3% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less of non-oleosome plant constituents. The obtained suspension can further be described as an oil-in- water (O / W) emulsion and is generally milky or creamy in appearance. The oleosome suspension can further, in some embodiments, be prepared to comprise small quantities, generally constituting 2% (w / w) or less, of ingredients to protect the suspension against undesirable alterations caused by physical, chemical or biological agents, especially when the preparation is stored for longer periods of time or exposed to temperature fluctuations. Thus, for example, small quantities of preservatives, and agents enhancing the physico- chemical or sensory properties of oleosomes can be included in the oleosome suspension. Stabilizing agents are generally selected to be suitable for inclusion in a dermal topical product. The oleosome suspension further can contain varying amounts of water, for example more than 10% and less than 65% water by volume, more preferably more than 15% and less than 50% water by volume, and most preferably more than 20% water by volume and less than 50% water by volume, as well as minor amounts of salts. Various suitable methods for the preparation of oleosome suspensions are known to the art and are further described in for example U.S. Pat. No.6,146,645, U.S. Pat. No.6,183,762, U.S. Pat. No.6,210,742, U.S. Pat. No.6,372,234, U.S. Pat. No.6,582,710, U.S. Pat. No.6,596,287, U.S. Pat. No.6,599,513, U.S. Pat. No.6,761,914, and U.S. Pat. No. 8,597,694, which are all incorporated herein by reference. The obtained oleosome suspensions, which may be obtained as herein described, constitute suitable oleosome preparations for use as an ingredient in accordance with the present disclosure. It is noted however the manner in which an oleosomepreparation suitable in accordance with the present disclosure is obtained is without particular restrictions and may be as desired, and the present disclosure is not limited by the process used to obtain a suitable oleosome preparation suspension.

[0133] As hereinbefore noted, in one preferred embodiment, a nucleic acid sequence encoding FGF-2 may be operably fused to a nucleic acid sequence encoding an oleosome associated polypeptide. In such embodiment, upon expression of an oleosome associated polypeptide-FGF-2 fusion polypeptide in a plant tissue comprising oleosome producing cells, for example, plant seed cells, the plant oleosomes within the plant seed cells comprise an oleosome associated polypeptide-FGF-2 fusion polypeptide. An oleosome suspension may be prepared from such plant tissue, using, for example, techniques as hereinbefore described, and since the oleosome associated polypeptide is embedded in the oleosomes, the FGF-2 polypeptide, fused to the oleosome associated polypeptide, is associated with the oleosomes within the oleosome suspension.

[0134] Thus, to briefly recap, example processes have been described that can be used to obtain a more or less pure FGF-2 polypeptide preparation, and a more or less pure oleosome preparation. These preparations may be used to prepare the dermal topical dermal formulations of the present disclosure. Oleosome-emulsified FGF-2

[0135] As hereinbefore noted, in accordance with one aspect hereof, the formulations of the present disclosure comprise oleosome-emulsified FGF-2. In one embodiment, initially, an initial preparatory formulation comprising oleosome-emulsified FGF-2 may be obtained by providing a more or less pure oleosome preparation and a more or less pure FGF-2 preparation, each of which may be prepared and provided using the example processes hereinbefore described. The FGF-2 preparation and oleosome preparation can be contacted with one another in a suitable vessel, for example, in a beaker, flask, or tank, and agitated, mixed, homogenized, or prepared therein under conditions sufficient to emulsify the FGF-2 polypeptide within the oleosome preparation. Mixing, agitation, homogenizing, and / or preparation conditionsmay be selected as desired but are generally preferably sufficiently gentle to maintain the structural integrity of the oleosomes, and sufficiently vigorous to obtain a homogenous preparation containing oleosome-emulsified FGF-2 polypeptide. Conditions generally deemed too vigorous are conditions which result in release of oil from the oleosomes in the preparation, as may be determined by visual inspection for the presence of oil droplets or a layer of oil in the preparation, while conditions deemed too gentle are generally conditions that fail to result in a forming a homogenous preparation containing oleosome- emulsified FGF-2 polypeptide. Suitable mixing conditions include, for example, gentle mixing or stirring using a mixer or a stirrer, at temperatures ranging from about 4 °C to about 60 °C, or from about room temperature to about 50 °C, for from at least about 5 minutes, and up to about 2 hours. Thus, for example, an FGF-2 polypeptide preparation may be mixed with an oleosome preparation at room temperature (22 ± 3 °C), or at a higher temperature, for example, 45 °C, and processes conducted under such conditions, relative to each other, may be referred to as cold and hot processes. The thus obtained preparation may be referred to as an initial preparatory formulation containing oleosome-emulsified FGF-2 polypeptide.

[0136] Optionally, following its constitution, the initial preparatory formulation containing oleosome-emulsified FGF-2 may be incubated at a temperature which may range from about 4 °C to about 60 °C, for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, inclusive, or for a period of 1 to 2 weeks, to allow the preparation to equilibrate.

[0137] Furthermore, in some embodiments, additional formulary ingredients may be included in the initial preparatory formulation containing oleosome-emulsified FGF-2, including agents that further facilitate association of the FGF-2 polypeptide with the oleosomes including, for example, a lipid droplet associated protein (LDAP), an acyl-CoA binding protein, a lipase, a lipoxygenase, or an antibody, for example, an antibody, for example, a bi- specific antibody with specificity for FGF-2 and an oleosome associated polypeptide, or agents that further facilitate emulsification of FGF-2, such as an emulsifier. These additional formulary ingredients may be included prior to the optional incubations.

[0138] Also further included in the initial preparatory formulation containing oleosome-emulsified FGF-2 may be small amounts of preservative agents, preferably dermatologically acceptable preservative agents, e.g., a benzoate salt, a sorbate (i.e., a salt or acid of sorbic acid, for example potassium sorbate), propionate (i.e., a salt or acid of propionic acid, for example sodium propionate), dihydroactetic acid, or a blend of gluconolactone and sodium benzoate (e.g., GeoGard Ultra®) up to 2%. Preservative formulary ingredients may be included prior to the optional incubations.

[0139] The concentration of FGF-2 in the initial preparatory formulation may vary but generally constitutes no more than 1% (w / w), no more than 0.5% (w / w), no more than 0.05% (w / w), no more than 0.005% (w / w), no more than 0.0005% (w / w), or no more than 0.00005% (w / w) of the initial preparatory formulation.

[0140] It is noted that, surprisingly, oleosome-emulsified FGF-2 polypeptide is stable, and preparations containing oleosome-emulsified FGF-2 polypeptide can, surprisingly, be stored at room temperature for at least 1 month, at least 3 months, at least 6 months, or at least 1 year without substantive loss of biological function of FGF-2.

[0141] As hereinbefore noted, in one preferred embodiment, a nucleic acid sequence encoding FGF-2 may be operably fused to a nucleic acid sequence encoding an oleosome associated polypeptide and expressed in oleosome containing plant tissues or parts, such as plant seeds. Oleosomes within an oleosome preparation obtained from such plant tissues or plant parts comprise an FGF-2 polypeptide, and the FGF-2 polypeptide can be said to be emulsified by the oleosomes within the oleosome preparation. Hence a more or less pure oleosome preparation obtained in accordance with such embodiment contains an oleosome-emulsified FGF-2 polypeptide. Dermal topical formulations comprising oleosome-emulsified FGF-2

[0142] According to some aspects, once an initial preparatory formulation comprising oleosome-emulsified FGF-2 has been obtained, the initial preparatory formulation may be used for dermal topical application. However, it is generally preferred to prepare a further finished dermal topical formulation comprising oleosome-emulsified FGF-2. In general, in this respect,in accordance with some aspects, the initial preparatory formulation is contacted with at least one other ingredient suitable for use in a dermal topical formulation, including notably a dermally acceptable diluent, carrier, or excipient (i.e., a substance included in a finished formulation which does not act as an active, for example, a vehicle or medium). The initial preparatory formulation and diluent, carrier, or excipient may be mixed, agitated, homogenized, or prepared, preferably until a homogenous mixture of the diluent, carrier, or excipient and the initial preparatory formulation comprising oleosome- emulsified FGF2 is obtained, and a dermal topical formulation comprising oleosome-emulsified FGF2 is formed, wherein such mixture is suitable for use as a formulation for dermal topical application, or, optionally, is suitable for further formulation using additional formulary ingredients. The diluent, carrier, or excipient may be any suitable dermally acceptable diluent, carrier, or excipient, and in one embodiment, is a diluent, carrier, or excipient not endogenously present in the plant or seed material used to obtain the oleosome preparation. Furthermore, the diluent, carrier, or excipient may be provided in any form, including, for example, as a solution, suspension, gel, liquid, solid, powder, or crystal. The quantity of the diluent, carrier, or excipient, or mixture thereof, may vary and depends on the type of finished dermal topical formulation that is prepared. Typically, the carrier, diluent, or excipient, or mixture thereof constitutes at least 0.5% or about 0.5% (w / w), at least 1% or about 1% (w / w), at least 5% or about 5% (w / w), at least 10% or about 10% (w / w), at least 20% or about 20% (w / w), at least 30% or about 30% (w / w), at least at least 40% or about 40% (w / w), at least 50% or about 50% (w / w), at least 60% or about 60% (w / w), at least 70% or about 70% (w / w), at least 80% or about 80% (w / w), at least 90% or about 90% (w / w), or at least 95% or about 95% (w / w), at least 99% or about 99% (w / w), at least 99.9% or about 99.9% (w / w) of a topical dermal formulation in accordance herewith, or from 0.5% or about 0.5% to 99.9% or about 99.9%, or from 1% or about 1% to 99.9% or about 99.9%, or from 5% or about 5% to 99.9% or about 99.9%, or from 10% or about 10% to 99.9% or about 99.9%, or from 20% or about 20% to 99.9% or about 99.9%, or from 30% or about 30% to 99.9% or about 99.9%, or from 40% or about 40% to 99.9% or about 99.9%, or from 50% or about 50% to 99.9% or about 99.9%, or from 60% or about 60% to 99.9% or about 99.9%, or from 70% or about 70% to 99.9% orabout 99.9%, or from 80% or about 80% to 99.9% or about 99.9%, or from 90% or about 90% to 99.9% or about 99.9% of a topical dermal formulation in accordance herewith.

[0143] Generally, a plurality of ingredients is provided, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ingredients, in addition to the initial preparatory formulation comprising oleosome-emulsified FGF-2, to prepare a finished dermal topical formulation. As noted, most preferably, one of these is at least, a dermally acceptable diluent, carrier, or excipient, or a mixture thereof. In embodiments hereof that include a plurality of formulary ingredients, such ingredients may be combined, mixed, agitated, and homogenized, sequentially or simultaneously. It will be understood that where ingredients are combined sequentially, one or more intermediate formulations may be formed, until a final formulary ingredient has been incorporated to form a finished dermal topical formulation.

[0144] In some embodiments, a formulation suitable for inclusion in a dermal topical formulation comprising a mixture of a plurality of ingredients is pre-formed, and the initial preparatory formulation comprising oleosome- emulsified FGF-2 is separately provided and incorporated in the pre-formed ingredient mixture.

[0145] In some embodiments, the initial preparatory formulation comprising oleosome-emulsified FGF-2 is incorporated during formulation. In such embodiments, the initial preparatory formulation comprising oleosome- emulsified FGF-2 may be added separately, or the initial preparatory formulation comprising oleosome-emulsified FGF-2 may be incorporated together with one or more other formulary ingredients, including, for example, a carrier, diluent, or excipient.

[0146] Thus, it is to be understood that in order to prepare a finished dermal topical formulation, the number of various formulary ingredients and order in which various formulary ingredients may be combined, including oleosome-emulsified FGF-2, a dermally acceptable carrier, diluent, or excipient, and optionally, other formulary ingredients, may be varied, and that two or more formulary ingredients may be pre-mixed before being combined with others to from an intermediate dermal topical formulation or a finished dermal topical formulation.

[0147] The final concentration of FGF-2 and oleosomes in the finished dermal topical formulation may vary. In some embodiments, the finished dermal formulation may comprise at least 0.00001% (w / w) or about 0.00001% (w / w) FGF-2. In other embodiments, the finished formulation may comprise at least 0.00005% or about 0.00005% (w / w), at least 0.0001% or about 0.0001% (w / w), at least 0.00015% or about 0.00015% (w / w), at least 0.00025% or about 0.00025% (w / w) FGF-2, inclusive of the dermal topical formulation. In some embodiments, the finished dermal topical formulation may comprise from 0.00001%, or about 0.00001% (w / w) to 1% or about 1% (w / w) FGF-2, or from 0.00005%, or about 0.00005% (w / w)to 0.5 % or about 0.5% (w / w) FGF-2, from 0.0001%, or about 0.0001% (w / w) to 0.0005% or about 0.0005% (w / w) FGF-2, or from 0.00015%, or about 0.00015% (w / w) to 0.00025% or about 0.00025% (w / w)FGF-2, inclusive of the dermal topical formulation. In some embodiments, the finished dermal topical formulation may comprise 0.00015% (w / w) or about 0.00015% (w / w), 0.00016% (w / w) or about 0.00016% (w / w), 0.00017% (w / w) or about 0.00017% (w / w), 0.00018% (w / w) or about 0.00018% (w / w), 0.00019% (w / w) or about 0.00019% (w / w), or 0.00020% (w / w) or about 0.00020% (w / w) FGF-2, inclusive of the dermal topical formulation.

[0148] In some embodiments, the finished dermal formulation may comprise at least from about 0.0001% (w / w) to about 95% (w / w) oleosomes. In some embodiments, the finished dermal formulation may comprise at least from about 5% (w / w) to about 95% (w / w) oleosomes. In other embodiments, the finished formulation may comprise at least 0.0001% or about 0.0001% (w / w), 0.005% or about 0.005% (w / w), at least 0.1% or about 0.1% (w / w), at least 1% or about 1% (w / w), at least 10% or about 10% (w / w), at least 20% or about 20% (w / w), at least 30% or about 30% (w / w), at least 40% or about 40% (w / w), at least 50% or about 50% (w / w), at least 60% or about 60% (w / w), at least 70% or about 70% (w / w), at least 80% or about 80% (w / w), at least 90% or about 90% (w / w), at least 95% or about 95% (w / w) oleosomes. In some embodiments, the finished formulation may comprise from 0.0001%, or about 0.0001% (w / w) to 0.05% oleosomes or about 0.05% (w / w) oleosomes, or from 0.005%, or about 0.005% (w / w) to 0.1% oleosomes or about 0.1% (w / w) oleosomes, or from 0.005%, or about 0.005% (w / w) to 0.6% oleosomes or about 0.6% (w / w) oleosomes, or from 0.01%, or about 0.01% (w / w) to 0.05% oleosomes or about0.05% (w / w) oleosomes, or from 0.0001%, or about 0.0001% (w / w) to 95% or about 95% (w / w) oleosomes. In some embodiments, the finished formulation may comprise from 5%, or about 5% (w / w) to 95% or about 95% oleosomes (w / w), or from 10%, or about 10% (w / w) to 90% or about 90% (w / w) oleosomes, from 10%, or about 10% (w / w) to 80% or about 80% (w / w) oleosomes, or from 10%, or about 10% (w / w) to 70% or about 70% (w / w) oleosomes, from 10%, or about 10% (w / w) to 60% or about 60% (w / w) oleosomes, from 10%, or about 10% (w / w) to 50% or about 50% (w / w) oleosomes, from 10%, or about 10% (w / w) to 40% or about 40% (w / w) oleosomes, from 10%, or about 10% (w / w) to 30% or about 30% (w / w) oleosomes, from 10%, or about 10% (w / w) to 20% or about 20% (w / w) oleosomes, or from 5%, or about 5% (w / w) to 10% or about 10% (w / w) oleosomes, inclusive of the dermal topical formulation. The concentrations FGF-2 of oleosomes in the finished formulation may be optimized or adjusted, for example by preparing a plurality of sample formulations, each including a different FGF-2 and / or oleosome concentration, applying each sample to, for example, an area of skin of a test subject, and evaluating the application for example with respect to, for example, skin aging or skin wound healing. Then, a formulation comprising a certain concentration FGF-2 and / or oleosomes may be selected that provides the most desirable effect.

[0149] In some embodiments, the diluent, carrier, or excipient, or mixtures thereof, incorporated in the dermal topical products of the present disclosure may be synthetic ingredients.

[0150] In some embodiments, the diluent, carrier, or excipient, or mixtures thereof, incorporated in the dermal topical products of the present disclosure may be natural ingredients.

[0151] Some optional excipients, diluent, carriers, and other formulary ingredients are described below in relation to techniques for formulation of dermal topical formulations.

[0152] In some embodiments, excipients included in the dermal topical formulations of the present disclosure can be white petrolatum (e.g., CAS No. 80009-03-8), cetyl alcohol (e.g., CAS No. 36653-82-4), polyethylene glycol (PEG), e.g., polyethylene glycol 400 (e.g., CAS No. 25322-68-3), or methylparaben (e.g., CAS No.99-76-3), or mixtures thereof.

[0153] In some embodiments, diluents included in the dermal topical formulations of the present disclosure can be water; a buffer, for example a phosphate buffer or a citrate buffer; a salt solution, for example, a sodium carbonate solution; or an organic solvent, for example, an alcohol, or mixtures thereof.

[0154] In some embodiments, carriers included in the dermal topical formulations of the present disclosure can be oils, for example, essential oils or lipids, to facilitate more even distribution of the oleosome-emulsified FGF2 onto the skin to which the dermal topical formulation is applied.

[0155] In some embodiments, additional formulary ingredients included in the dermal topical formulations of the present disclosure can be moisturizers. In some embodiments, the moisturizers can be natural compounds, including, for example, glycerin, sodium pyrrolidone carboxylic acid (PCA), honey, or Aloe leaf extract, or mixtures thereof. In other embodiments, the moisturizers can be synthetic compounds, including, for example, propylene glycol.

[0156] In some embodiments, additional formulary ingredients included in the dermal topical formulations of the present disclosure can be emollients, including in some embodiments, oils, waxes, lipids, or other water insoluble compounds, or mixtures thereof. It is noted that oils and lipids may be incorporated in the formulations of the present disclosure by using exogenous oils and lipids. In some embodiments, emollients that may be included in the dermal topical formulations of the present disclosure are natural compounds, including, for example, Brassica oil, cetyl alcohol, shea butter, safflower oil, sunflower oil, oleyl lactate, dicaprylyl ether, beeswax, carnauba wax or mixtures thereof; and / or synthetic compounds, including, for example, dimethicone, cyclopentasiloxane or C12-15 alkyl benzoate, or mixtures thereof.

[0157] In some embodiments, other formulary ingredients incorporated in the dermal topical formulations of the present disclosure may be surface active agents, including, for example, cationic surfactants, including, for example, natural cationic surfactants, such as brassicyl isoleucinate esylate; and / or synthetic cationic surfactants, such as cetrimonium chloride and behentrimonium, chloride, or mixtures thereof; including further, for example, anionic surfactants, including, for example, natural anionic surfactants, such as sodium coco-sulfate; and / or synthetic anionic surfactants, such as sodiumlaureth sulfate, or mixtures thereof; including further, for example, non-ionic surfactants, including for example, natural non-ionic surfactants, such as sorbitan olivate or sorbitan oleate, or mixtures thereof; and / or synthetic non- ionic surfactants, such as polysorbate 20 and PEG-100 stearate, or mixtures thereof; and including, still further, zwitterionic surfactants, including, for example, natural zwitterionic surfactants, such as decyl glucoside or lauryl glucoside; and / or synthetic zwitterionic surfactants, such as sodium cocoamphoacetate, or mixtures thereof.

[0158] In some embodiments, other formulary ingredients incorporated in the dermal topical formulations of the present disclosure are viscosity- modulating agents. In some embodiments, the viscosity modulating agents are natural compounds, including, for example, xanthan gum, carrageenan gum, sclerotium gum, Brassica alcohol, cellulose or cellulose derivatives, or mixtures thereof. In other embodiments, the viscosity-modulating agents can be synthetic agents, including, for example, carbomer, sodium acrylate copolymer or cetyl alcohol, or mixtures thereof.

[0159] In some embodiments, other formulary ingredients that can be incorporated in the dermal topical formulations include chelating agents. In some embodiments, the chelating agents can be natural chelating agents, such as sodium gluconate. In other embodiments, the chelating agents can be synthetic chelating agents, such as disodium EDTA.

[0160] In some embodiments, other formulary ingredients that can be included in the dermal topical formulations of the present disclosure, are small amounts of preservative agents, e.g., a benzoate salt, a sorbate (i.e., a salt or acid of sorbic acid, for example potassium sorbate), propionate (i.e., a salt or acid of propionic acid, for example sodium propionate), dihydroactetic acid, or a blend of gluconolactone and sodium benzoate (e.g., GeoGard Ultra®), or mixtures thereof, preferably, in amounts less than 2% (w / w).

[0161] In some embodiments, other formulary ingredients incorporated in the dermal topical formulations of the present disclosure can be active ingredients, in addition to FGF-2. In some embodiments, the active ingredients can be natural active ingredients, including, for example, bisabolol, shea butter unsaponifiables, tocopherol, a natural sunscreen agent, or rosemary extract, or mixtures thereof. In other embodiments, the active ingredients can be syntheticactive ingredients including, for example, palmitoyl tetrapeptide-7, polyquaternium-10, a synthetic sunscreen agent, or mixtures thereof.

[0162] The final concentrations of the diluents, excipients, carriers, and other formulary ingredients in the dermal topical formulations, the pH of the dermal topical formulations, the viscosity of the dermal topical formulations, and other chemical and physicochemical properties of the dermal topical formulations of the present disclosure, and the manner in which the personal care formulations are constituted may vary substantially depending on the desired use and performance characteristics of the dermal topical formulation. Those of skill in the art will be familiar with a variety of different methodologies and techniques, for example, heating methodologies, stirring or mixing techniques, pH adjustment techniques, viscosity adjustment methodologies, and the like, all of which may be used, adjusted and / or optimized in to prepare suitable dermal topical formulations.

[0163] In some embodiments, a dermal topical formulation prepared in accordance herewith can be a cream, a crème, a gel, a foam, an ointment, a paste, a lotion, a serum a tincture, a milk, a fluid, or a powder.

[0164] In some embodiments, a dermal topical formulation prepared in accordance with the present disclosure can be a skin care formulation, including without limitation a skin cream, a crème, a facial cream, an eye cream, a skin cleanser, a day cream, a skin toner, a lotion, a serum, a facial mask, an anti- aging cream, an anti-wrinkle cream, a cold weather cream, a foot cream, or a hand cream.

[0165] In some embodiments, a dermal topical formulation prepared in accordance with the present disclosure, can be a bath and body formulation, including a body wash, a bar soap, a bath gel, or a shower gel.

[0166] In some embodiments, a dermal topical formulation prepared in accordance with the present disclosure can be a lip care product, including a lip balm, lip moisturizer, lip conditioner, or lipstick.

[0167] In some embodiments, a dermal topical formulation prepared in accordance herewith can be a make-up formulation, including, for example, a foundation, a mascara, a blush, bronzer, eye shadow, a nail polish or a make- up remover.Treatment of skin conditions with formulations comprising oleosome- emulsified FGF-2

[0168] The dermal topical formulations of the present disclosure may be used to prevent, treat, or ameliorate a variety of skin or dermatological conditions, notably, in particular, skin or dermatological conditions, which are associated with a lack of dermal collagen biosynthesis. In this respect, it has been found by the present inventors that, surprisingly, the formulations of the present disclosure stabilize FGF-2, and allow for FGF-2 to penetrate in the deeper dermal layers of the skin of a subject, including a human subject, to which the formulation is applied, and in doing so can cross the stratum corneum. Furthermore, the formulations of the present disclosure allow retention of the biological activity of FGF-2, and upon topical administration the topical dermal formulations can effect in vivo activation of collagen biosynthesis (including Type I collagen) in the dermal layers of the skin of a subject.

[0169] Furthermore, the dermal topical formulations of the present disclosure can stimulate fibroblast cell proliferation. In this respect, skin fibroblast cells, are cells that are known to be implicated in wound healing, including in the breakdown of the fibrin clot, creating new extracellular matrix (ECM), and collagen structures to support other cells participating in wound healing.

[0170] The dermal topical formulations of the present disclosure can generally be applied to the exterior surface area of a subject, including a human subject, or optionally, an animal, such as a dog or a cat. Generally, the dermal topical formulations can be administered by topically applying a sufficient quantity, for example, a dose of about 1 to 10 grams of a dermal topical formulation containing from about 0.00001% (w / w) to about 0.5% (w / w), or 0.0001% (w / w) to about 0.0005% (w / w), or 0.00015% (w / w) to about 0.00025% (w / w), FGF-2 polypeptide to a skin area selected to be treated, for example, a skin area of from about 1 cm2to about 20 cm2. The formulation can generally be manually applied by gently rubbing a dose of the dermal topical formulation onto the selected skin area for a sufficient period of time to allow the formulation to spread more or less evenly across the selected skin area. The formulation can be self-administered or it can be administered to a subject by a health practitioner. Upon administration of the dermal topical formulation, theoleosome-emulsified FGF-2 polypeptide can, surprisingly, diminish the barrier properties of the skin, and traverse the skin’s outermost barrier, notably the stratum corneum. Furthermore, surprisingly, when administered in the formulations of the present disclosure, FGF-2 peptide can remain biologically active, and it can stimulate collagen production in the dermal layers of the skin. As will be understood by those of skill in the art, treatments may be iterative, for example, once daily, two times daily, or three times daily, for a certain period of time, for example, for a period of 1 week, 2 weeks, 3 weeks, or 4 weeks, or any other period that results in successful treatment of a skin condition.

[0171] In some embodiments, wherein the FGF-2 polypeptide is produced as a fusion polypeptide between an oleosome associated polypeptide and the FGF-2 polypeptide and the oleosome associated polypeptide and FGF- 2 polypeptide are separated by a cleavable polypeptide linker, the cleavable polypeptide linker may be cleaved by endogenous proteases present in the skin of the subject to whom the dermal topical formulation is administered. Such cleavage may then result in the in vivo separation of the FGF-2 polypeptide from the oleosome associated polypeptide.

[0172] In some embodiments, the skin condition that can be treated is a condition associated with skin aging. In this respect, the dermal topical formulations of the present disclosure may, for example, be used to prevent treat, or ameliorate skin aging spots, wrinkles, pigmentation, loss of skin tone, rough skin texture, uneven skin texture, broken capillaries, redness, weak skin barrier, and blotchiness.

[0173] In some embodiments, the skin condition can be a condition associated with skin injury, for example an acute wound, such as a superficial burn injury, a deep burn injury, skin trauma, or a surgical incision wound, or the skin injury can be associated with a non-injury caused dermatological condition, such as alopecia, psoriasis, atopic dermatitis, acne, or a diabetic foot ulcer, for example.

[0174] The above disclosure generally describes various aspects of methods and compositions of the present disclosure. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitutionof equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. SUMMARY OF SEQUENCES

[0175] SEQ.ID NO: 1 and SEQ.ID NO: 2 set forth a nucleic acid sequence encoding fibroblast growth factor 2 (FGF-2) (encoding mature polypeptide; codon optimized for expression in Camelina sativa), and the amino acid sequence of fibroblast growth factor 2 (FGF-2) (mature polypeptide) encoded by SEQ.ID NO: 1, respectively.

[0176] SEQ.ID NO: 3 and SEQ.ID NO: 4 set forth a nucleic acid sequence encoding fibroblast growth factor 2 (FGF-2) (including pro-polypeptide; codon optimized for expression in Camelina sativa), and the amino acid sequence of fibroblast growth factor 2 (FGF-2) (including pro-polypeptide) encoded by SEQ.ID NO: 3, respectively.

[0177] SEQ.ID NO: 5 and SEQ.ID NO: 6 set forth a nucleic acid sequence encoding an Arabidopsis thaliana oleosin, and the amino acid sequence of an oleosin encoded by SEQ.ID NO: 5, respectively.

[0178] SEQ.ID NO: 7 and SEQ.ID NO: 8 set forth a nucleic acid sequence encoding a Camelina sativa oleosin, and the amino acid sequence of an oleosin encoded by SEQ.ID NO: 7, respectively.

[0179] SEQ.ID NO: 9 and SEQ.ID NO: 10 set forth a nucleic acid sequence encoding another Camelina sativa oleosin, and the amino acid sequence of an oleosin encoded by SEQ.ID NO: 9, respectively.

[0180] SEQ.ID NO: 11 and SEQ.ID NO: 12 set forth a nucleic acid sequence encoding another Camelina sativa oleosin, and the amino acid sequence of an oleosin encoded by SEQ.ID NO: 11, respectively.

[0181] SEQ.ID NO: 13 and SEQ.ID NO: 14 set forth a nucleic acid sequence encoding a Camelina sativa caleosin, and the amino acid sequence of a caleosin encoded by SEQ.ID NO: 13, respectively.

[0182] SEQ.ID NO: 15 and SEQ.ID NO: 16 set forth a nucleic acid sequence encoding another Camelina sativa caleosin, and the amino acid sequence of a caleosin encoded by SEQ.ID NO: 15, respectively.

[0183] SEQ.ID NO: 17 and SEQ.ID NO: 18 set forth a nucleic acid sequence encoding another Camelina sativa caleosin, and the amino acid sequence of a caleosin encoded by SEQ.ID NO: 17, respectively.

[0184] SEQ.ID NO: 19 and SEQ.ID NO: 20 set forth a nucleic acid sequence encoding a steroleosin, and the amino acid sequence of a steroleosin encoded by SEQ.ID NO: 19, respectively.

[0185] SEQ.ID NO: 21 and SEQ.ID NO: 22 set forth a nucleic acid sequence of an expression construct encoding an oleosin-prochymosin-human FGF-2, and the oleosin-prochymosin-human FGF-2 encoded by SEQ.ID NO: 21, respectively.

[0186] SEQ.ID NO: 23 and SEQ.ID NO: 24 set forth a nucleic acid sequence of an expression construct encoding an oleosin-human FGF-2 , and the oleosin-human FGF-2 encoded by SEQ.ID NO: 23, respectively.

[0187] SEQ.ID NO: 25 and SEQ.ID NO: 26 set forth a nucleic acid sequence encoding a prochymosin polypeptide linker sequence, prochymosin polypeptide linker encoded by SEQ.ID NO: 25, respectively. EXAMPLES Example 1 – Preparation of a basic dermal topical formulation containing oleosome-emulsified FGF-2.

[0188] For blending, rhFGF-2 bearing oleosomes from transformed Camelina sativa seed expressing Arabidopsis 18kDa oleosin:Prochymosin maturation cleavage site:human Fibroblast Growth Factor 2 (Oleo:ProChym:hFGF2) fusion protein (SEQ.ID NO: 22) are added and mixed with water, glycerin USP, Xanthan gum and preservatives including Glucolactone and Na Benzoate to a final percentage of 1.5 and 0.5%, respectively (adjusted to account for the preservatives in the oleosomes- rhFGH-2 preparatory formulation). The preparation of rhFGF-2 bearing oleosomes from transgenic Camelina sativa seed is described in Example 10. The blend is prepared by first mixing under high-speed Glycerin USP (5% weight) and Xanthan gum (2% weight). The glycerin / xanthan gum is added to Water (~85% weight) under strong agitation, creating a vortex when adding the water, and mixed for 30 minutes. Next the preservatives (final combination 2%)are added to the gel under low shear and mixed well. Finally, the oleosomes- rhFGH-2 (0.6% (w / w) are added and mixed until the gel is completely homogenous. At this point the pH is measured, and adjusted if needed, to ensure the pH <5.0, and a blended (formulated) preparation is obtained. The obtained basic oleosome-rhFGF-2 preparation may be used as a basic dermal topical formulation, but it may also be used as an ingredient for formulating more complex dermal topical formulations, including, for example, the topical dermal formulations described in Examples 6, 7, and 8.

[0189] The gel is then dispensed to receiving HDPE containers sealed and stored under temperature-controlled conditions (room temperature). Example 2 – Stimulation of dermal collagen production by dermal topical formulations containing oleosome-emulsified FGF-2.

[0190] MatTek’s EpiDermFT system consists of normal, human epidermal keratinocytes and normal, human dermal fibroblasts cultured to form a multilayered model of the human dermis and epidermis. Cultured at the air- liquid interface in wall-to-wall tissue culture inserts, EpiDermFT attains a level of differentiation and structural characteristics to simulate human skin and is organized with keratin 5 expressing basal cells, involucrin and keratin 10 expressing spinous and granular layers, and cornified epidermal layers analogous to those found in vivo. The epidermal and dermal layers are mitotically and metabolically active and exhibit in vivo-like morphological and growth characteristics which are uniform and highly reproducible.

[0191] For testing the unblended engineered oleosome emulsions bearing rhEGF (Oleo:CLS:rhEGF), MatTek EpiDermFT was treated topically, at two doses, with test materials for 96 hours, with a media change every 48 hours. At the end of the treatment period changes in Type I collagen were measured in tissue homogenates and tissue culture media using ELISA (see: FIG.1).

[0192] For testing the blended (formulated) engineered oleosome emulsions bearing rhFGF-2 (blended as described in Example 1), MatTek EpiDermFT was treated topically with test materials, at three different doses, for 96 hours, with a media change every 48 hours. At the end of the treatment period changes in Type I collagen was measured in tissue homogenates and tissue culture media using ELISA (see: FIG.2).

[0193] For the Type I Collagen ELISA, a series of type I C-peptide standards were prepared ranging from 0 ng / ml to 640 ng / ml. Tissue homogenates or collected cell culture media (at 0-48 hours and 48-96 hours) was plated and incubated with peroxidase-labeled anti-procollagen type I-C peptide antibody. After the incubation the wells were aspirated and washed three times with wash buffer. After the last wash, peroxidase substrate solution (hydrogen peroxide and tetramethylbenzidine as a chromagen) was added to each well and the plate was incubated at room temperature. After incubating, the reaction was stopped with 1N sulfuric acid and plates were read at 450 nm.

[0194] To quantify the amount of type I collagen (COL1) in the tissue and culture media at the two time points, absorbance of test articles was compared to the COL1 standard curve (regression analysis to establish best fit line).

[0195] When topically applied to the surface, a significant increase (*; ANOVA p=0.05) in COL1 was measured in response to the presence of blended rhFGFP emulsions and unblended rhEGF emulsions in MatTek’s EpiDermFT system when compared to untreated tissue (see: FIG. 1). In addition, the blended rhFGF2 emulsion in hydrated gel, also resulted in a significant increase in COL1 in the 3D ex vivo skin model, compared to untreated tissue. Example 3 – Stability of dermal topical formulations containing oleosome- emulsified FGF-2.

[0196] To test the stability, engineered oleosome emulsions expressing Oleo-CLS-rhFGF2 were purified and stored in buffer. Subsequently, the rhFGF2 was quantified in order to test stability in a 3T3 fibroblast cellular proliferation assay against an equivalent concentration of commercially sourced rhFGF2 (see: FIG.3A). Commercially sourced rhFGF2 (Thermo Fisher Scientific® Inc.) was reconstituted in the same buffer. Both buffered Oleo-CLS-rhFGF2 emulsions and commercial rhFGF2 were stored at 4oC for 5 weeks and retested for 3T3 cellular proliferation using the same concentrations of samples tested at time zero. In this comparison the ability of commercially rhFGF2 to stimulate 3T3 cells was completely obliterated, whereas the engineered oleosome emulsions bearing the rhFGF2 retained activity as measured by 3T3 cellular proliferation at all concentrations tested.

[0197] In a second test, the stability of the Oleo-CLS-rhFGF2 emulsion in comparison to commercially sourced rhFGF2 was determined from 3T3 cell culture media at different time points as measured by Western Blotting (see: FIG.3B). In this study the commercially soured rhFGF2 was only 25% of the original concentration in the media at 12 hours and was no longer present after 24 hours. In comparison the Oleo-CLS-rhFGF2 half-life was 3X longer in culture media at 37oC.

[0198] Similarly, a second set of Western Blots comparing the presence or reduction of commercially sourced rhFGF2 and Oleo-CLS-rhFGF2 was evaluated over 72 hours in comparison to non-engineered oleosomes (Purified wild type (native oleosomes) and irrelevant protein (Newborn Calf Serum) control (see: FIG.3C). Example 4 – Stability of dermal topical formulations containing oleosome- emulsified FGF-2.

[0199] To test the stability of Engineered oleosomes bearing rhFGF2 fusion proteins, extracted oleosomes were stored at room temperature and the integrity of the fusion protein followed over time in comparison to a commercially sourced rhFGF2 over time. The integrity of the fusion protein was assessed by quantifying the presence of the fusion protein (resolving at the expected molecular weight and absent in wild type oleosomes) using SDS-PAGE under reducing conditions. FIG. 4 shows the integrity of the fusion protein in the extracted oleosome samples and the subsequent quantification over time for samples stored at ambient temperature. The fusion protein in these conditions was within 5% of the original sample for up to 12 months, whereas the commercially sourced rhFGF2 degraded to less than 25% within 1 month, and slowly degraded thereafter to below 20% after 12 months.

[0200] For blended samples the integrity of the oleosome preparations were followed using laser diffraction to ascertain the oleosome size of samples stored at ambient (room) temperature or under accelerated conditions (40oC). Protein content was also monitored over time using the intensity resolved marker bands of the Coomassie stained SDS-PAGE. Table 1 demonstrates that the integrity of oleosomes and protein profiles was maintained under room temperature or accelerated conditions suggesting the oleosomes remainedintact (did not degrade or coalescence) and thereby the covalently attached fusion proteins remained anchored to the extracted oleosomes formulated in the blend.

[0201] Table 1: results obtained in the performance of experiments assaying the integrity of blended (formulated) oleosomes comprising FGF-2 and epidermal growth factor (EGF) over time (0 to 12 months) at 40oC and room temperature. Oleosin- Stability Analysis Time (months) fusion condition 0 0.5 1 3 6 12 FGF2 RT Oil body 3.63 3.72 3.73 3.61 TBD TBD size [µm]* Protein 0.15 0.16 0.13 1.2 TBD TBD content [%]** AC Oil body 3.63 3.69 3.69 4.33 TBD TBD size [µm] Protein 0.15 0.14 0.14 0.87 TBD TBD content [%] EGF RT Oil body 4.10 4.12 4.12 4.17 TBD TBD size [µm] Protein 0.13 0.12 0.15 0.5 TBD TBD content [%] AC Oil body 4.10 4.07 4.15 4.04 TBD TBD size [µm]Protein 0.13 0.12 0.17 0.93 TBD TBD content [%] * as determined by laser diffraction; ** as determined by Coomassie stained SDS-PAGE Example 5 – Treatment of skin conditions using dermal topical formulations containing oleosome-emulsified FGF-2.

[0202] To evaluate the efficacy of engineered oleosomes in vivo, gel creams consisting of rhFGF-2 bearing oleosomes and / or rhEGF oleosomes that have been blended to simulate a skin care hydrated gel at 5% and or 1% were tested on healthy human volunteers as part of a double-blinded observational study designed to evaluate anti-aging, firming and depigmenting efficacy. In the blended gels at 1% and additional analysis of barrier effect was determined.

[0203] For 5% blend, a hydrated gel consisting of 5% blend (from Example 1) was mixed with components to represent a hydrated gel that was preserved and pH adjusted and aliquoted to HDPE containers to provide each subject with enough materials to apply twice daily for the 28 day period of the study. Similarly, for 1% blends, a hydrated gel consisting of 1% blend (from Example 1) was mixed in the same composition and aliquoted in same containers.

[0204] Each treatment and placebo groups consisted of 20 recruited healthy volunteers, using predefined inclusion criteria and approved methods. The study was conducted under consent by the volunteers in accordance with privacy laws and ethics approval by the GLP testing facility. For the studies, the blended oleosomes were applied to areas of the face at either 5% or 1%, twice daily (morning and night) for 28 days. Baseline measurements were conducted at the start of the study (Day 0; T0) and measurements performed on Day 14 and 28 (T14 and T28, respectively) of the study. For evaluation of anti-wrinkle efficacy, the micro-relief of the skin (primary and secondary lines), was measured using Visio3D dermaTOP BLUE system (EOTECH) for arithmetical mean of roughness (Ra) and mean depth of roughness (Rz). For pigmentation, the melanin content of dark spots on each volunteer in the test and placebogroups was measured using a Mexameter® MX18. Skin firmness and elasticity analysis (R0 and R2) were assessed using a Cutometer® (Courage-Khazaka GmbH, Germany). For blends used at 1%, barrier effect (trans epidermal water loss (TEWL)) was assessed by Tewameter® TM300 at the start of the treatment (T0) and after 14 (T14) and 28 (T28) days of daily application. To ensure reliable and accurate reproducibility, the same area of skin is analyzed for each subject at the different time points using proband’s positioning together with alignment of 3D software, based on Bruekchmann’s program OPTOCAT.

[0205] In regard to anti-wrinkle properties (Table 2A), the 5% blend containing oleosomes bearing rhFGF2, significant decreases in Ra and Rz were observed in comparison to T0 at 28 days after treatment. In comparison, the placebo had no significant effect. For the corresponding 1% blend, a significant decreases in Ra was observed at day 28. There was also a decrease in Rz, but it was not statistically significant. For the 1% blend containing oleosomes bearing rhEGF, a significant decrease in Ra was also observed at day 28. Together these results demonstrate both rhFGF2 and rhEGF can significantly reduce age-related wrinkles on skin after treatment for 28 days.

[0206] In regard to depigmentation (Table 2B), the 5% blend containing oleosomes bearing rhFGF2, significantly decreased melanin content in the application area after 28 days treatment. At 1% blend usage, both oleosomes bearing rhFGF2 and rhEGF, significantly decreased melanin content in the application area after both 14- and 28-days treatment. In comparison the placebo had no significant effects in both 5% and 1% studies. Together these results demonstrate both rhFGF2 and rhEGF can significantly reduce age related spots on the skin after treatment.

[0207] In regard to skin firmness (Table 2C), the 5% blend containing oleosomes bearing rhFGF2, significantly increased after 28 days treatment as measured by R0 parameter. In addition, a corresponding significant increase in skin elasticity as measured by the R2 parameter after 28 days treatment. At 1% blend usage, both oleosomes bearing rhFGF2 and rhEGF significantly increased skin firmness as measured by R0 parameter after both 14- and 28- days treatment. In comparison the placebo had no significant effects in either study. Together these results demonstrate both rhFGF2 and rhEGF cansignificantly increase skin firmness and at higher dosing or perhaps prolonged treatment can significantly increase skin elasticity.

[0208] In regard to skin barrier effects (Table 2D), blends containing oleosomes bearing rhFGF2 or rhEGF significantly decreased skin TEWL at both 14- and 28- days treatment, whereas the placebo had no effect. Together these results demonstrate both rhFGF2 and rhEGF can prevent skin moisture loss following treatment.

[0209] Tables 2A, 2B, 2C and 2D: results obtained in the performance of experiments assaying certain skin aging parameters: in-vivo anti-wrinkle efficacy using a 5% oleosome-emulsified FGF-2 formulation and a 1% oleosome-emulsified FGF-2 formulation over time (t = 0 days (“T0”), 14 days (“T14”), and 28 days (“T28”)) compared against a placebo (Table 2A); in-vivo depigmentation efficacy using a 5% oleosome- emulsified FGF-2 formulation and a 1% oleosome-emulsified FGF-2 formulation over time (t = 0 days (“T0”), 14 days (“T14”), and 28 days (“T28”)) compared against a placebo (Table 2B); in-vivo firming (R0 value) and elasticity (R2 value) using a 5% oleosome- emulsified FGF-2 formulation and a 1% oleosome-emulsified FGF-2 formulation over time (t = 0 days (“T0”), 14 days (“T14”), and 28 days (“T28”)) compared against a placebo (Table 2C); and in-vivo trans epidermal water loss (TEWL) using a 1% oleosome- emulsified FGF-2 formulation and a 1% oleosome- emulsified EGF formulation over time (t = 0 days (“T0”), 14 days (“T14”), and 28 days (“T28”)) compared against a placebo (Table 2D).

[0210] Table 2A Treatment Time Mean Ra % Signif Mean Rz % Signif (days (mm) + / - Variatio icanc (mm) + / - Variatio icanc ) SD n vs T0 e SD n vs T0 e Placebo T0 0.059 + / - N / A N / A 0.243 + / - N / A N / A 0.017 0.136 T14 0.059 + / - 0.8% ns 0.247 + / - 1.8% ns 0.017 0.140T28 0.059 + / - -0.4% ns 0.247 + / - 1.8% Ns 0.017 0.145 5% T0 0.062 + / - N / A N / A 0.269 + / - N / A N / A rhFGF2 0.015 0.114 T14 0.061 + / - -1.2% ns 0.264 + / - -1.8% Ns 0.013 0.104 T28 0.058 + / - -6.5% * 0.248 + / - -7.6% * 0.013 0.098 Placebo T0 0.0625 N / A N / A 0.2428 N / A N / A + / - + / - 0.0141 0.0580 T14 0.0614 -1.8% ns 0.2430 0.1% ns + / - + / - 0.0169 0.0663 T28 0.0606 -3.0% ns 0.2383 -1.8% ns + / - + / - 0.0137 0.0623 1% T0 0.0643 N / A N / A 0.2382 N / A N / A rhFGF2 + / - + / - 0.0199 0.0691 T14 0.0605 -5.8% ns 0.2277 -4.4% ns + / - + / - 0.0167 0.0562 T28 0.0610 -5.2% * 0.2316 -2.8% Ns + / - + / - 0.0185 0.0628 1% rhEGF T0 0.0681 N / A N / A 0.2536 N / A N / A + / - + / - 0.0194 0.0660 T14 0.0639 -6.1% ns 0.2372 -6.5% ns + / - + / - 0.0183 0.0632T28 0.0630 -7.5% * 0.2370 -6.5% ns + / - + / - 0.0162 0.0528

[0211] Table 2B Treatment Time (days) Mean Melanin % Variation vs T0 Significance value + / -SD Placebo T0 224.4 + / - 109.3 N / A N / A T14 224.6 + / - 114.5 0.1% ns T28 230.6 + / - 112.6 2.8% ns 5% rhFGF2 T0 216.0 + / - 66.1 N / A N / A T14 213.9 + / - 68.8 -1.0% ns T28 207.2 + / - 64.9 -4.1% * Placebo T0 197.7 + / - 49.3 N / A N / A T14 198.0 + / - 56.2 0.1% ns T28 201.7 + / - 51.3 2.0% ns 1% rhFGF2 T0 190.4 + / - 35.0 N / A N / A T14 162.3 + / - 36.8 -14.8% *** T28 156.1 + / - 36.2 -18.0% **** 1% rhEGF T0 201.0 + / - 44.9 N / A N / A T14 179.9 + / - 45.6 -10.5% *T28 169.5 + / - 39.2 -15.7% **

[0212] Table 2C Treatment Time Mean R0 % Signifi Mean R2 % Signifi (days) + / -SD Variat cance + / -SD Variat cance ion ion vs T0 vs T0 Placebo T0 0.328 + / - N / A N / A 0.512 + / - N / A N / A 0.086 0.117 T14 0.339 + / - 3.1% ns 0.505 + / - -1.2% ns 0.069 0.116 T28 0.298 + / - -9.2% ns 0.504 + / - -1.4% ns 0.057 0.107 5% T0 0.319 + / - N / A N / A 0.481 + / - N / A N / A rhFGF2 0.070 0.101 T14 0.309 + / - -3.0% ns 0.485 + / - 0.8% ns 0.055 0.104 T28 0.270 + / - - *** 0.510 + / - 6.0% * 0.045 15.3 0.090 % Placebo T0 0.153 + / - N / A N / A 0.504 + / - N / A N / A 0.025 0.086 T14 0.149 + / - -2.5% ns 0.497 + / - -1.5% ns 0.024 0.117 T28 0.157 + / - 2.6% ns 0.503 + / - -0.1% ns 0.030 0.096 1% T0 0.202 + / - N / A N / A 0.495 + / - N / A N / A rhFGF2 0.030 0.078 T14 0.160 + / - - *** 0.506 + / - 2.1% ns 0.049 21.0 0.091 %T28 0.174 + / - - ** 0.509 + / - 2.8% ns 0.037 14.0 0.090 % 1% rhEGF T0 0.185 + / - N / A N / A 0.504 + / - N / A N / A 0.040 0.073 T14 0.145 + / - - *** 0.500 + / - -0.9% ns 0.029 21.8 0.081 % T28 0.158 + / - - * 0.517 + / - 2.5% ns 0.035 14.4 0.068 %

[0213] Table 2D Treatment Time Mean TEWL + / - % Variation vs T0 Significance (days) SD Placebo T0 17.5 + / - 6.7 N / A N / A T14 16.7 + / - 6.4 -4.5% ns T28 16.6 + / - 7.8 -5.1% ns 1% rhFGF2 T0 17.3 + / - 3.5 N / A N / A T14 14.6 + / - 2.1 -15.5% ** T28 14.4 + / - 2.6 -16.9% *** 1% rhEGF T0 18.1 + / - 3.8 N / A N / A T14 16.1 + / - 3.7 -10.8% * T28 15.1 + / - 2.9 -16.6% **Example 6 – Preparation of an anti-aging eye cream formulation containing oleosome-emulsified FGF-2.

[0214] An anti-aging eye cream formulation for topical dermal application was prepared in accordance with the following formulary and process.

[0215] Table 3 (Formulary, Eye Cream) No Ingredient (supplier) INCI Name % (w / w) 1 Water Aqua63.802 Glycerin Glycerin 3.75 Keltrol-CG-SFT (CP 3 Xanthan Gum 0.50 Kelco) Floraester K20W 4 Aqua (and) Hydrolyzed Jojoba Esters1.25(Cargill) 5 Olivem 1000 (Hallstar) Cetearyl / Olivate (and) Sorbitan Olivate5.50Cetyl Palmitate (and) Sorbitan 6 Oliwax LC (Hallstar) 2.00 Palmitate (and) Sorbitan Olivate 7 Cetiol OE (BASF) Dicaprylyl Ether7.508 Cetiol (LC (BASF) Coco-Caprylate / Caprate7.509 Corviox T-70 (BASF) Tocopherol 0.20 Benzyl Alcohol (and) Salicylic Acid 10 Geogard ECT (Arxada) 1.00 (and) Glycerin (and) Sorbic Acid Glycerin (and) Aqua (and) Spirulina 11 Aquacell (Barnett) 2.00 Platensis Extract Basic Oleosomes-FGF- 2 Ingredient (prepared 12N.A5.00as described in.Example 1)

[0216] Referring to Table 3, ingredient No.2 and Ingredient No.3 were weighed and mixed together in a stainless steel mixing tank containing a side scraper to obtain a homogenous mixture of ingredients No. 2 and No. 3. Ingredient No.1 was then added to the mixture, and mixed with ingredient No.1, strongly agitating the mixture for 30 minutes. Thereafter ingredient No.4 wasadded and the temperature of the mixture was increased to 800C, while mixing. The obtained homogenous mixture is referred to as the Phase A mixture.

[0217] Ingredient Nos.5 – 9 were then weighed and heated in another stainless steel mixing tank and heated to 800C while mixing to homogeneity. The obtained homogenous mixture is referred to as the Phase B mixture.

[0218] The Phase B mixture was then added to the Phase A mixture and homogenized for 10 minutes. Thereafter the obtained homogenous mixture was cooled using the side scraper in the stainless steel mixing tank.

[0219] When the mixture reached a temperature of 450C, ingredient Nos. 10 and 11 were added while continuing to mix.

[0220] When the mixture reached a temperature of 400C, ingredient No 12 was added while continuing to mix. The temperature of the formulation was then cooled to room temperature to obtain a homogenously constituted anti- aging eye cream formulation.

[0221] The pH of the anti-aging eye cream topical formulation was 6.5 and the viscosity 25,000 centipoise (cps). Example 7 – Preparation of a rejuvenating serum formulation containing oleosome-emulsified FGF-2.

[0222] A rejuvenating serum formulation for topical dermal application was prepared in accordance with the following formulary and process.

[0223] Table 4 (Formulary, Serum) No Ingredient (supplier) INCI Name % (w / w) 1 Water Aqua 73.00 Gluconolactone (and) Sodium 2 Geogard Ultra (Arxada) 1.00 Benzoate 3 Propanediol Propanediol 5.00 Xanthan Gum (and) Lecithin (and) 4 Siligel (Lucas Meyer) 1.00 Sclerotium Gum (and) Pullulan Glycerin (and) Glycine Soja (Soybean) 5 Lysofix (Lucas Meyer)_ 4.00 Seed ExtractGlyceryl Stearate Citrate (and) Heliofeel 22 MB (Lucas 6 Polycglyceryl-3-Stearate (and) 2.00 Meyer) Hydrogenated Lecithin Grankane 1214-LC Coconit alkanes (and) Coco- 7 6.00 (Grant) Caprylate / Caprate VAS-Vegetable Hydrogenated Ethylhexyl Olivate (and) 8 Alternative to Silicone Hydrogenated Olive oil 6.00 (EFP Biotek) Unsaponifiables Water (and) Glycerin (and) Xanthan Gum (and) Camelina Sativa Peauvita (Core Oleosomes / Camelina Sativa sr- 9 1.00 Biogenesis) (Arabidopsis Thaliana polypeptide-2 sh-Polypeptide-1 (and) Gluconolactone 10 Basic Oleosomes-FGF- 2 Ingredient (prepared N.A. 1.00 as described in Example 1)

[0224] Referring to Table 4, ingredient No. 1 was weighed and mixed with ingredient in a stainless steel mixing tank until ingredient No. 2 was dissolved. There after ingredient No.3 and No.4 were premixed and added to the ingredient No.1 / No.2 mixture and mixed for 5 minutes. Ingredient No. 5 was then added and the obtained mixture containing ingredient Nos.1 – 5 was mixed for 5 more minutes. The obtained homogenous mixture is referred to as the Phase A mixture.

[0225] Ingredient Nos.6 – 8 were then weighed and heated in another stainless steel mixing tank and mixed. The obtained homogenous mixture is referred to as the Phase B mixture.

[0226] The Phase A and Phase B mixture were then each heated to 750C, and the Phase B mixture was added to the Phase A mixture, mixing at high speed for 10 minutes. Thereafter the obtained homogenous mixture cooled using the side scraper in the stainless steel mixing tank.

[0227] When the mixture reached a temperature of 400C, ingredient Nos. 9 and 10 were consecutively added while continuing to mix. The temperature ofthe formulation was then cooled to room temperature to obtain a homogenously constituted rejuvenation serum formulation.

[0228] The pH of the rejuvenation serum formulation was 4.5. Example 8 – Preparation of a rejuvenating crème formulation containing oleosome-emulsified FGF-2.

[0229] A rejuvenation crème formulation for topical dermal application was prepared in accordance with the following formulary and process.

[0230] Table 5 (Formulary, Crème) No Ingredient (supplier) INCI Name % (w / w) 1 Water Aqua 68.00 Gluconolactone (and) Sodium 2 Geogard Ultra (Arxada) 1.00 Benzoate 3 Propanediol Propanediol 3.00 Keltrol-CG-SFT (CP 4 Xanthan Gum 0.50 Kelco) Glyceryl Stearate Citrate (and) Heliofeel 22 MB (Lucas 5 Polycglyceryl-3-Stearate (and) 4.00 Meyer) Hydrogenated Lecithin 6 Olivem 900 (Hallstar) Sorbitan Olivate 2.00 Ultra Refined Shea 7 Butyrospermum Parkii Butter 5.00 Butter Ultra Refined 8 Deodorized Cocoa Theobroma Cacao Seed Butter5.00Butter Behenyl Behenate (and) Kester Wax-K- BWR 9 polyhydroxystearic Acid (and) 3.00 (Koster Keunen) Euphorbia Cerfira (Candilla) Wax 10 Golden Jojoba Oil Simmondsia Chinensis Seed Oil 3.00 Grankane 1214-LC Coconit alkanes (and) Coco- 11 3.00 (Grant) Caprylate / Caprate 12 Vitamin E Tocopherol 0.50 Peauvita (Core Water (and) Glycerin (and) Xanthan 13 1.00 Biogenesis) Gum (and) Camelina SativaOleosomes / Camelina Sativa sr- (Arabidopsis Thaliana polypeptide-2 sh-Polypeptide-1 (and) Gluconolactone Basic Oleosomes-FGF- 2 Ingredient (prepared 141.00as described inN.A.Example 1)

[0231] Referring to Table 5, ingredient No. 1 was weighed and mixed with ingredient in a stainless steel mixing tank until ingredient No. 2 was dissolved. Thereafter ingredient No.3 and No.4 were weighed premixed and added to the ingredient No. 1 / No. 2 mixture and mixed for 5 minutes. The obtained homogenous mixture is referred to as the Phase A mixture.

[0232] Ingredient Nos.6 - 11 were then weighed and heated in another stainless steel mixing tank and mixed to homogeneity. The obtained mixture is referred to as the Phase B mixture.

[0233] The Phase A and Phase B mixture were then each heated to 800C, and the Phase B mixture was added to the Phase A mixture, mixing at high speed for 10 minutes. Thereafter the obtained homogenous mixture was cooled using the side scraper in the stainless steel mixing tank.

[0234] When the mixture reached a temperature of 400C, ingredient Nos. 12 - 14 were consecutively added while continuing to mix. The temperature of the formulation was then cooled to room temperature to obtain a homogenously constituted rejuvenation crème formulation.

[0235] The pH of the rejuvenation crème formulation was 4.5. Example 9 – Expression of oleosin-FGF-2 fusion polypeptide in Camelina sativa.

[0236] A chimeric gene construct containing a human FGF-2 gene having SEQ.ID NO: 1 was genetically fused to the C-terminal end of an Arabidopsis thaliana oleosin gene (SEQ.ID NO: 5), and the two sequences were either separated by a sequence encoding a prochymosin sequence (SEQ.ID NO: 25) or directly fused. The entire nucleic acid sequences are set forth inSEQ.ID NO: 21 (including prochymosin sequence) and SEQ.ID NO: 23 (directly fused), and the amino acid sequence of the polypeptide encoded by the same is set forth in SEQ.ID NO: 22 (including prochymosin sequence) and SEQ.ID NO: 24 (directly fused). Furthermore, an Arabidopsis thaliana oleosin promoter was included in the chimeric construct to drive expression. The entire construct, including the fusion gene and Arabidopsis oleosin promoter was cloned into an Agrobacterium plant transformation vector containing a DsRed (red fluorescent protein) for screening and screening and selection and a nos terminator sequence, and the vector was then used to transform Agrobacterium cells.

[0237] To transform Camelina plants, a floral dip method was used. Briefly, Camelina sativa plants at an early flowering stage were dipped into a suspension containing the transformed Agrobacterium cells. Camelina sativa plants were grown, and T1 seeds were collected and germinated on selective media (i.e., media containing the herbicide corresponding with the herbicide resistance marker). T2 seeds were subsequently analyzed for the presence of the oleosin-FGF2 fusion gene and polypeptide by polymerase chain reaction (PCR), quantitative reverse transcription PCR (qRT-PCR), and Western blotting. Transgenic Camelina sativa seeds showing the presence of oleosin- FGF2 fusion gene and polypeptide (SEQ.ID NO: 22) were then used for growing further plant generations, and to bulk up transgenic Camelina sativa seed quantities comprising the oleosin-FGF2 fusion gene. Example 10 – Recovery of oleosome-emulsified FGF-2 from transgenic Camelina sativa seed.

[0238] Plant seeds from transgenic Camelina sativa plants (obtained as described in Example 9) were harvested when the seeds had fully matured. One kilogram Camelina sativa seeds was ground using a ball mill in the presence of 4 liters tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.5). The obtained homogenate was then subjected to low speed centrifugation (5,000 x g for 10 minutes). The oleosome fraction contained in the top layer of the obtained centrifugate was collected and resuspended in Tris buffer (pH 7.5). Upon resuspension the oleosome fraction was centrifuged again (5,000 x g for 10 minutes), and thereafter collected. This washing step was repeated two additional times. The thus recovered oleosome fraction can be said to be a moreor less pure oleosome fraction and may be evaluated for the presence of FGF- 2, for example, by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and Western blotting using anti-FGF-2 antibodies.

Claims

CLAIMS 1. A dermal topical formulation comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to stimulate dermal collagen production, together with a dermally acceptable diluent, excipient, or carrier, or mixture thereof.

2. The dermal topical formulation according to claim 1, wherein the FGF-2 polypeptide is fused to an oleosome associated polypeptide, the FGF-2 polypeptide and the oleosome associated polypeptide forming a single contiguous polypeptide.

3. The dermal topical formulation according to claim 1, wherein the FGF-2 polypeptide is fused to an oleosome associated polypeptide, wherein the FGF- 2 polypeptide and the oleosome associated polypeptide are separated by a cleavable polypeptide linker, the oleosome associated polypeptide, the cleavable polypeptide linker, and the FGF-2 polypeptide forming a single contiguous polypeptide.

4. The dermal topical formulation according to claim 2 or 3, wherein the oleosome associated protein is an oleosin, a caleosin, or a steroleosin.

5. The dermal topical formulation according to any one of claims 1 to 4, wherein the FGF-2 polypeptide constitutes from about 0.00005% (w / w) up to about 0.5% (w / w) of the formulation.

6. The dermal topical formulation according to any one of claims 1 to 4, wherein the FGF-2 polypeptide constitutes from about 0.0001% (w / w) up to about 0.0005% (w / w) of the formulation.

7. The dermal topical formulation according to any one of claims 1 to 4, wherein the FGF-2 polypeptide constitutes from about 0.00015% (w / w) up to about 0.00025% (w / w) of the formulation.

8. The dermal topical formulation according to any one of claims 1 to 4, wherein the formulation contains from about 0.0001% (w / w) up to about 10% (w / w) oleosomes.

9. The dermal topical formulation according to any one of claims 1 to 4, wherein the formulation contains from about 0.005% (w / w) up to about 0.6% (w / w) oleosomes.

10. The dermal topical formulation according to any one of claims 1 to 4, wherein the formulation contains from about 0.01% (w / w) up to about 0.05% (w / w) oleosomes.

11. The dermal topical formulation according to any one of claims 1 to 4, wherein the formulation contains from about 90% (w / w) up to about 99.1% (w / w) of the diluent, excipient, or carrier, or mixture thereof.

12. The dermal topical formulation according to any one of claims 1 to 11, wherein the FGF-2 polypeptide is emulsified by a plant oleosome obtained from the group of plant species consisting of almond (Prunus dulcis), camelina (Camelina sativa), hemp (Cannabis sativa), linseed / flax (Linum usitatissimum), mustard (Brassica spp. and Sinapis alba), rapeseed (Brassica spp.), safflower (Carthamus tinctorius), and sunflower (Helianthus annuus).

13. The dermal topical formulation according to any one of claims 1 to 11, wherein the FGF-2 polypeptide is emulsified by a plant oleosome obtained from a plant belonging to the plant genus Camelina.

14. The dermal topical formulation according to any one of claims 1 to 11, wherein the FGF-2 polypeptide is emulsified by a plant oleosome obtained from a plant belonging to the plant species Camelina sativa.

15. The dermal topical formulation according to any one of claims 1 to 14, wherein the dermal topical formulation further comprises a preservative agent.

16. The dermal topical formulation according to any one of claims 1 to 15, wherein the dermal topical formulation further comprises at least one of anadditional formulary ingredient selected from a moisturizer, an emollient, a surfactant, a viscosity modifying agent, or a chelating agent.

17. The dermal topical formulation according to any one of claims 1 to 16, wherein the dermal topical formulation is a cream, a crème, a gel, a foam, an ointment, a paste, a lotion, serum, a tincture, a milk, a fluid, or a powder.

18. The dermal topical formulation according to any one of claims 1 to 17, for use in the prevention, amelioration, or treatment of a skin condition in a subject in need thereof.

19. The dermal topical formulation for use according to claim 18, wherein the skin condition is a condition associated with skin aging.

20. The dermal topical formulation for use according to claim 19, wherein the skin condition associated with skin aging is selected from skin aging spots, wrinkles, pigmentation, loss of skin tone, rough skin texture, uneven skin texture, broken capillaries, redness, weak skin barrier, and blotchiness.

21. The dermal topical formulation for use according to claim 18, wherein the skin condition is a condition associated with skin injury.

22. The dermal topical formulation for use according to claim 21, wherein the dermal topical formulation stimulates skin fibroblast cell proliferation to thereby effect healing of injured skin.

23. The dermal topical formulation for use according to claim 21, wherein the skin condition associated with skin injury is an acute wound.

24. The dermal topical formulation for use according to claim 23, wherein the acute wound is a superficial burn injury, a deep burn injury, skin trauma, or a surgical incision wound.

25. The dermal topical formulation for use according to claim 21, wherein the skin condition associated with skin injury is alopecia, psoriasis, atopic dermatitis, acne, or a diabetic foot ulcer.

26. The dermal topical formulation for use according to any one of claims 18 to 25 wherein the subject is a human subject.

27. A method of making a dermal topical formulation according to any one of claims 1 to 17, the method comprising: providing oleosome-emulsified FGF-2; providing a dermally acceptable diluent, excipient, or carrier, or mixture thereof; and contacting the oleosome-emulsified FGF-2 with the dermally acceptable diluent, excipient, or carrier to prepare a dermal topical formulation comprising a sufficient quantity of an oleosome-emulsified fibroblast growth factor 2 (FGF-2) polypeptide to stimulate dermal collagen production.

28. The method according to claim 27, wherein the FGF-2 polypeptide is fused to an oleosome associated polypeptide, the FGF-2 polypeptide and the oleosome associated polypeptide forming a single contiguous polypeptide.

29. The method according to claim 27, wherein the FGF-2 polypeptide is fused to an oleosome associated polypeptide, wherein the FGF-2 polypeptide and the oleosome associated polypeptide are separated by a cleavable polypeptide linker, the oleosome associated polypeptide, the cleavable polypeptide linker, and the FGF-2 polypeptide forming a single contiguous polypeptide.

30. The method according to claim 28 or 29, wherein the oleosome associated protein is an oleosin, a caleosin, or a steroleosin.

31. The method according to any one of claims 27 to 30, wherein the FGF-2 polypeptide constitutes from about 0.00005% (w / w) up to about 0.5% (w / w) of the formulation.

32. The method according to any one of claims 27 to 30, wherein the FGF-2 polypeptide constitutes from about 0.0001% (w / w) up to about 0.0005% (w / w) of the formulation.

33. The method according to any one of claims 27 to 30, wherein the FGF-2 polypeptide constitutes from about 0.00015% (w / w) up to about 0.00025% (w / w) of the formulation.

34. The method according to any one of claims 27 to 30, wherein the formulation contains from about 0.0001% (w / w) up to about 10% (w / w) oleosomes.

35. The method according to any one of claims 27 to 30, wherein the formulation contains from about 0.005% (w / w) up to about 0.6% (w / w) oleosomes 36. The method according to any one of claims 27 to 30, wherein the formulation can contain from about 0.01% (w / w) up to about 0.05% (w / w) oleosomes.

37. The method according to any one of claims 27 to 30, wherein the formulation contains from about 90% (w / w) up to about 99.9% (w / w) of the diluent, excipient, or carrier, or mixture thereof.

38. The method according to any one of claims 27 to 37, wherein the FGF-2 polypeptide is emulsified by a plant oleosome obtained from the group of plant species consisting of almond (Prunus dulcis), camelina (Camelina sativa), hemp (Cannabis sativa), linseed / flax (Linum usitatissimum), mustard (Brassica spp. and Sinapis alba), rapeseed (Brassica spp., including, without limitation, the canola genotype), safflower (Carthamus tinctorius), and sunflower (Helianthus annuus).

39. The method according to any one of claims 27 to 37, wherein the FGF-2 polypeptide is emulsified by a plant oleosome obtained from a plant belonging to the plant genus Camelina.

40. The method according to any one of claims 27 to 37, wherein the FGF-2 polypeptide is emulsified by a plant oleosome obtained from a plant belonging to the plant species Camelina sativa.

41. The method according to any one of claims 27 to 40, wherein the dermal topical formulation further comprises a preservative agent.

42. The method according to any one of claims 27 to 40, wherein the dermal topical formulation further comprises at least one of an additional formulary ingredient selected from a moisturizer, an emollient, a surfactant, a viscosity modifying agent, or a chelating agent.

43. The method according to any one of claims 27 to 42, wherein the dermal topical formulation is a cream, a crème, a gel, a foam, an ointment, a paste, a lotion, a serum, a tincture, a milk, a fluid, or a powder.

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