Crosslinked materials

Abstract

To provide the synthesis, formulation and uses of crosslinkable entities and crosslinked materials.SOLUTION: The present disclosure provides a variety of insights relating to provision of crosslinked materials (e.g., gels or other high molecular weight materials) in or on a target site of interest (e.g., in situ). Among other things, the present disclosure provides an insight that lipophilicity of a crosslinkable entity (e.g., comprising a polymer moiety and a crosslink moiety) can be tuned to achieve a desired rate and / or extent of penetration after application to a site (e.g., on a skin surface or otherwise to a skin site above a target site of interest).SELECTED DRAWING: None

Description

[Background technology]

[0001] background Various methods for the introduction or production of crosslinked materials at or on a particular site of interest, such as at the site of surgical or traumatic destruction (e.g., at the site of an organ, connective tissue, muscle, tendon, and / or membrane; see, e.g., Seal et al. Mater Sci. Eng 34:147, 2001). Various systems have been developed, some of which have shown promise, for example, to effectively seal internal wounds and / or achieve tissue joining for improved wound healing (see, e.g., Ruel-Gariepy et al. Eur. J. Pharm. Biopharm. 58:409, 2004). [Prior art documents] [Non-patent literature]

[0002] [Non-Patent Document 1] Seal et al. Mater Sci. Eng34:147, 2001 [Non-Patent Document 2] Ruel-Gariepy et al. Eur. J.Pharm. Biopharm. 58:409, 2004 Summary of the Invention [Means for solving the problem]

[0003] Abstract The present disclosure provides various insights regarding providing a crosslinked material (eg, a gel or other high molecular weight material) in or on a target site of interest (eg, in situ). In particular, the present disclosure provides the insight that the lipophilicity of a crosslinkable entity (e.g., including a polymeric portion and a crosslinking portion) can be adjusted to achieve a desired rate and / or extent of penetration following application to a site (e.g., to a skin site on the skin surface or otherwise above a target site of interest).

[0004] Alternatively or additionally, the present disclosure provides the insight that low viscosity preparations of crosslinkable entities have desirable characteristics for certain embodiments, e.g., allowing the crosslinked materials they produce to better fill spaces and / or structures therein (e.g., microcavities on a surface) and / or to better contact tissues and / or structures thereover.

[0005] Alternatively or additionally, the present disclosure provides the insight that a crosslinked material as provided by the present disclosure may exhibit improved durability / persistence compared to its initial crosslinkable entity in the relevant biological system (e.g., separate from and / or prior to their crosslinking to produce the relevant crosslinked material).

[0006] Still further, the present disclosure provides insight that it may be beneficial and / or otherwise desirable in certain embodiments for the first crosslinkable entity and the second crosslinkable entity to be kept separate (i.e., chemically, physically and / or spatially) upon administration (e.g., during administration).

[0007] In particular, the present disclosure provides an embodiment in which one or more of a set of crosslinkable entities that crosslink with each other to produce a crosslinked material are characterized by a degree of lipophilicity as described herein.The present disclosure also provides an embodiment in which one or more crosslinkable entities are utilized as a low-viscosity preparation.The present disclosure also provides an embodiment in which the individual crosslinkable entities are maintained separate from each other upon administration (e.g., until and / or during administration).The present disclosure further provides insight into combinations of such embodiments, and into specific circumstances in which one or more of such embodiments may be particularly useful or effective.

[0008] In particular, the insights provided enable the selection and / or utilization of crosslinkable entities or components (e.g., polymers and / or crosslinking moieties), or combinations thereof, not previously contemplated or utilized for in situ crosslinking systems, and / or provide new methodologies for administering crosslinkable entities to achieve in situ crosslinked materials.

[0009] In some embodiments, the present disclosure provides a system including components that can be used together to provide an in situ crosslinked material. In some embodiments, the system provided includes a first crosslinkable entity and a second crosslinkable entity, at least one of which (and in many embodiments, both of which) has a lipophilicity as described herein, and the first and second entities crosslink with each other to form a crosslinked material (e.g., a gel) at or on a site of interest (e.g., in situ) having material and / or functional characteristics as described herein.

[0010] In some embodiments, the present disclosure provides methods of making and / or using such provided systems and / or components thereof.

[0011] In some provided methods, the first crosslinkable entity and the second crosslinkable entity are administered separately and sequentially to a site (e.g., on a skin surface), and in some such embodiments, the first crosslinkable entity is administered first and the second crosslinkable entity is administered a period of time later.

[0012] Also provided are various devices and techniques for storing and / or administering the first and / or second crosslinkable entity. In some embodiments, the provided devices maintain the first and second crosslinkable entities in physically separate locations, compartments, and / or containers. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows a portion of human skin including the epidermis and dermis.

[0014] [Diagram 2] FIG. 2 shows the 1H NMR of HA-gly-CBT in D2O of Example 1.

[0015] [Diagram 3] Figure 3 shows gel permeation chromatography (GPC) analysis of modified HA. Refractive index readings of unmodified HA (with average molecular weights of 10 kDa, 50 kDa, and 250 kDa) before conjugation are shown in Figure 3A, and UV absorbance readings (326 nm) of modified HA (HA-gly-CBT, with average molecular weights of 10 kDa, 50 kDa, and 250 kDa) are shown in Figure 3B.

[0016] [Figure 4] FIG. 4 shows the chemical structures of certain derivatives of CBT and HA (A and B, respectively).

[0017] [Diagram 5] FIG. 5 shows the 1H NMR of Cys-PEG-Cys in deuterated acetone of Example 1.

[0018] [Figure 6] FIG. 6 shows the 1H NMR of Cys-EDA-Cys in deuterated acetone of Example 1.

[0019] [Figure 7] 7 depicts an exemplary packaging of a first crosslinkable entity and a second crosslinkable entity that are dynamically blended at the time of use. The packaging shown shows that the two entities are mixed in a chamber immediately prior to dispensing.

[0020] [Figure 8] FIG. 8 depicts an exemplary packaging of a first crosslinkable entity and a second crosslinkable entity that can be individually dispensed.

[0021] [Figure 9] Figure 9 shows the resistance of cross-linked HA-gly-CBT to degradation by hyaluronidase. The plot in Figure 9A illustrates the fraction of HA-gly-CBT that remains as an insoluble gel and high molecular weight polymer after incubation with hyaluronidase for both cross-linked and non-cross-linked materials. Figure 9B is an image of a cross-linked HA-gly-CBT gel after incubation with hyaluronidase for 24 hours.

[0022] [Figure 10] FIG. 10 shows bright field and fluorescent (DAPI filter) images of pig skin sections after topical application of PBS (A) and HA-gly-CBT (B) to intact pig skin and after topical application of HA-gly-CBT (C) to tape-stripped pig skin.

[0023] [Figure 11]Figure 11 shows GPC analysis of skin extracts following topical application of HA-gly-CBT. Figure 11A shows a comparison of the GPC signal from HA-gly-CBT with the GPC signal measured for stratum corneum (SC) extracts and epidermis / dermis extracts, averaged over six samples. Figure 11B shows the average GPC signal for the SC extracts with associated error bars. Figure 11C shows the average GPC signal for the epidermis / dermis extracts with associated error bars.

[0024] [Figure 12] Figure 12 shows bright field and fluorescent (DAPI filter) images of pig skin sections after injection with HA-gly-CBT (A) or HA-gly-CBT followed by injection with Cys-PEG-Cys (B). Both sections were imaged directly after being sectioned and washed to extract soluble HA-gly-CBT.

[0025] [Figure 13] FIG. 13 depicts photographs of the skin after two days of application of the provided system compared to baseline and placebo application, showing improved skin smoothness.

[0026] [Figure 14] Figure 14 shows bright field and fluorescence (Cy5 filter) images of human skin sections following topical application of PBS (A) and AlexaFluor 647 tagged HA-gly-CBT (B and C) to disrupted human skin. The skin was tape stripped prior to application in one sample (B) and treated with a dermaroller containing 500 μm needles in another (C).

[0027] [Figure 15] FIG. 15 depicts exemplary administration sites for Example 6.

[0028] [Figure 16] FIG. 16 represents images at time points following administration of the system as described in Example 6.

[0029] [Figure 17] FIG. 17 represents biopsy images following administration of the system as described in Example 7.

[0030] [Figure 18] Figure 18 shows the local delivery of HA-gly-CBT (50 kDa, tagged with IR dye) into human skin treated with a dermaroller containing 500 μm needles. Figure 18A shows a fluorescent microscopy image of a skin sample obtained using a Cy5 filter. Figure 18B shows quantitative data of HA-gly-CBT to the SC, epidermis, and dermis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] definition About: The term "about" when used herein in reference to a value refers to a value that is similar in the context of the referenced value. In general, a person skilled in the art familiar with the context will recognize the relevant degree of variation that is encompassed by "about" in the context. For example, in some embodiments, the term "about" can encompass values ​​within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0032] Analog: As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity with the reference substance, e.g., shares a core or consensus structure, but also differs in certain distinct respects. In some embodiments, an analog is a substance that can be produced from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by performing a synthetic method that is substantially similar to (e.g., shares multiple steps with) the method that produces the reference substance. In some embodiments, an analog is produced or can be produced by performing a synthetic method that is different from the method used to produce the reference substance.

[0033] Associated: As used herein, the term "associated" refers to two events or entities that are "associated" with each other if the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with each other if they directly or indirectly interact with each other and thus are physically close to each other and / or remain close to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other, and in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other but are non-covalently associated, for example, by using hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0034] Biocompatible: The term "biocompatible," as used herein, refers to a material that, when placed in contact with living tissue, e.g., in vivo, does not cause significant harm to such tissue. In certain embodiments, materials are "biocompatible" if they are not toxic to cells. In certain embodiments, materials are biocompatible if their addition to cells in vitro results in 20% or less cell death and / or are biocompatible if their addition to cells in vitro results in 20% or less cell death and / or are biocompatible if their addition to cells in vivo ... They are "biocompatible" if their administration in vivo does not induce significant inflammation or other such adverse effects.

[0035] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, situations, sets of conditions, etc. that may not be identical to each other, but are similar enough to allow comparison between them, and thus one of skill in the art will recognize that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, equivalent sets of conditions, environments, individuals, or populations are characterized by multiple substantially identical features and one or a few altered features. One of skill in the art will understand what degree of uniqueness is required for two or more such sets of agents, entities, situations, conditions, etc. to be considered equivalent in any given environment, depending on the circumstances. For example, one of skill in the art will recognize that sets of environments, individuals, or populations are equivalent to each other when they are characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of environments, individuals, or populations are caused by or indicate variations in the altered features.

[0036] Corresponding to: As used herein in the context of polypeptides, nucleic acids, and compounds, the term "corresponding to" designates the location / identity of a structural element, e.g., an amino acid residue, a nucleotide residue, or a chemical moiety, in a compound or composition by comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, one of skill in the art will recognize that for simplicity, a residue in a polymer may be designated using a standard numbering system based on the relevant reference polymer, and thus, for example, a residue "corresponding to" a residue at position 190 of a reference polymer does not necessarily actually be the 190th residue in the polymer of interest, but rather refers to the residue equivalent to the residue found at position 190 in the reference polymer. One of skill in the art will readily recognize how to identify a "corresponding" residue in a polymer (e.g., using commercially available sequence comparison software for polypeptide and nucleic acid polymers, and manually as necessary for other polymers).

[0037] Designed: As used herein, the term "designed" refers to an agent that (i) has a structure that is selected or has been selected by the hand of man, (ii) is produced by a process that requires the hand of man, and / or (iii) is distinct from natural substances and other known agents.

[0038] Dosage form: Those skilled in the art will recognize that the term "dosage form" may be used to refer to a physically separate unit of an agent (e.g., a therapeutic, diagnostic, or cosmetic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of the agent. In some embodiments, such an amount is a unit dosage (or a whole fraction thereof) suitable for administration according to a dosing regimen that has been determined to correlate with a desired or beneficial (e.g., therapeutic and / or cosmetic) outcome when administered to a relevant population (i.e., using a therapeutic dosing regimen). In some embodiments, such an amount is a unit dosage (or a whole fraction thereof) suitable for administration according to a regimen that has been determined to correlate with a desired or beneficial cosmetic outcome (e.g., providing visible and / or tactile improvement to the skin) when administered to a relevant population. Those skilled in the art will recognize that the total amount of a composition or agent to be administered to a particular subject will be determined by one or more professionals (e.g., physicians, nurses, or other qualified professionals) responsible and may include administration of multiple dosage forms. In some embodiments, the dosage form may be a cream, gel, liquid, lotion, mist, mask, matrix, particle, paste, patch, powder, serum, solid, spray, or the like. The composition may be provided in a formulation that is or contains the compound (or a collection thereof), or a combination thereof.

[0039] Dosing regimen: Those skilled in the art will recognize that the term "dosing regimen" can be used to refer to a set of unit doses (typically two or more) that are administered individually to a subject, typically separated by a period of time. In some embodiments, a given drug has a recommended dosing regimen that can include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, the individual doses are separated from each other by periods of the same length, and in some embodiments, a dosing regimen includes multiple doses, and at least two different periods that separate the individual doses. In some embodiments, all doses in a dosing regimen are doses of the same unit dosage. In some embodiments, different doses in a dosing regimen are doses of different amounts. In some embodiments, a dosing regimen includes a first dose of a first dosage followed by one or more additional doses of a second dosage that is different from the first dosage. In some embodiments, the dosing regimen comprises a first dose of a first dosage followed by one or more additional doses of a second dosage that is the same as the first dosage. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population.

[0040] Excipient: As used herein, excipient refers to an inert agent (e.g., not a therapeutically active agent, such as a cosmetically active agent) that may be included in a pharmaceutical composition to, for example, provide or contribute to a desired consistency or stabilizing effect.

[0041] "Improve", "Enhance", "Inhibit" or "Reduce": As used herein, the terms "improve", "enhance", "inhibit" or "reduce", or grammatical equivalents thereof, refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in a comparable system known or predicted to respond in a particular manner in the presence of the relevant agent or treatment.

[0042] In situ: As used herein, the term "in situ" refers to a location at, in, or on a target site. For example, in some embodiments, a crosslinkable entity crosslinks in situ when it reacts to form a crosslinked material at, in, or on a target site of interest, e.g., on a tissue surface or within a tissue, and in many embodiments, the tissue of interest is skin.

[0043] Isolated: Isolated, as used herein, refers to substances and / or entities that (1) have been separated from at least some of the components with which they were associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) have been designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance can be considered to be "isolated" or even "pure" even after it has been combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the isolation or purity percentage of the substance is calculated without including such carriers or excipients.

[0044] Linker: Linker, as used herein, is used to refer to the exact portion of a multi-element drug that connects the different elements to one another.

[0045] Marker: As used herein, marker refers to an entity or moiety whose presence or level is characteristic of a particular condition or event. In some embodiments, the presence or level of a particular marker may be characteristic of the presence, state, or stage of a disease, disorder, or condition.

[0046] Subject: As used herein, the term "subject" refers to any living organism to which the provided system is or can be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Exemplary subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the subject is a human. In some embodiments, the subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, the subject exhibits one or more symptoms of a disorder or condition. In some embodiments, the subject has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or the presence of one or more tumors. In some embodiments, the subject is undergoing or has undergone a certain treatment to diagnose and / or treat a disease, disorder, or condition. In some embodiments, the subject refers to a human seeking cosmetic benefits and / or improvements, such as improving the appearance and / or feel of the skin.

[0047] Physiological conditions: Physiological conditions, as used herein, has its art-understood meaning referring to the conditions in which a cell or organism lives and / or reproduces. In some embodiments, the term refers to the conditions of the external or internal environment that may occur naturally for an organism or cellular system. In some embodiments, physiological conditions are conditions present inside the human or non-human animal body, particularly at and / or within a target site of interest. Physiological conditions typically include, for example, one or more of a temperature in the range of 20-40° C. (particularly about 37° C.), atmospheric pressure 1, pH 6-8, a glucose concentration of 1-20 mM, an oxygen concentration at atmospheric levels, and gravity as encountered on earth.

[0048] Reference: Reference, as used herein, describes a standard or control to which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally incorporated into a tangible medium. Typically, as can be understood by those skilled in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those under assessment. Those skilled in the art will recognize when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0049] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell, or an organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as the context will make clear, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). Such a "processed sample" can include, for example, material extracted from the sample or material obtained by subjecting the primary sample to one or more techniques, such as chromatography, extraction, precipitation, etc.

[0050] Substantial structural similarity: As used herein, the term "substantial structural similarity" refers to the presence of shared structural features at specific positions. In some embodiments, the term "substantial structural similarity" refers to the presence and / or uniqueness of structural elements, such as loops, sheets, helices, H-bond donors, H-bond acceptors, glycosylation patterns, salt bridges, disulfide bonds, and combinations thereof. In some embodiments, the term "substantial structural similarity" refers to the three-dimensional arrangement and / or orientation of atoms or moieties relative to each other (e.g., the distances and / or angles between or within the agent of interest and the reference agent).

[0051] Therapeutic Agent: As used herein, the phrase "therapeutic agent" generally refers to any agent that induces a desired pharmacological effect (which in some embodiments may be or may include a cosmetic effect) when administered to an organism. In some embodiments, an agent is considered to show an effect (i.e., is a therapeutic agent) if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, the appropriate population may be defined by certain criteria, such as a certain age group, sex, genetic background, pre-existing clinical conditions, etc., or combinations thereof. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, mitigate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent is a therapeutic agent that achieves a cosmetic effect (i.e., is a cosmetic agent). In some embodiments, a therapeutic agent can be used to improve the appearance and / or feel of the skin and / or achieve another cosmetic benefit.

[0052] Treat: As used herein, the terms "treat", "treatment" or "treating" refer to partial or complete relief, remission, delay in onset, inhibition, prevention, alleviation, and / or reduction in incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition, or achieving another desired physiological effect (e.g., a desired cosmetic effect, e.g., improvement in the appearance and / or feel of the skin, e.g., visible and / or tactile improvement in the skin). In some embodiments, treatment includes administration of an agent that produces a physiological effect. In some embodiments, treatment includes cosmetic treatments that, upon administration, improve the physical appearance in the manner described herein. In some embodiments, treatment can be administered to subjects who do not exhibit signs or features of a disease, disorder, and / or condition (e.g., can be prophylactic). In some embodiments, treatment can be administered to subjects who exhibit very early or mild signs or features of a disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who show established, severe, and / or end-stage symptoms of a disease, disorder, or condition.

[0053] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Crosslinkable entities The present disclosure provides certain techniques relating to the administration of crosslinkable entities to a subject, and in particular to the in situ crosslinking of such crosslinkable entities at, in or on a target site, which may be, for example, in or on the skin, e.g., a site on, in, or beneath the epidermis, dermis or underlying subcutaneous tissue.

[0054] In some embodiments, the present disclosure provides techniques for administering a system comprising a first crosslinkable entity and a second crosslinkable entity selected and / or designed to achieve the formation of a crosslinked material in situ. Those skilled in the art reading this disclosure will recognize that such crosslinked materials are typically characterized by one or more different physical properties, e.g., rheology, and / or chemical properties, e.g., molecular weight, compared to their parent crosslinkable entities. In some embodiments, such different properties may be or include, for example, improved durability / persistence in situ and / or in an otherwise relevant biological system.

[0055] As described herein, such first and second crosslinkable entities are characterized by their ability, when contacted with one another, to react with one another to form a crosslinked material in situ, e.g., without the administration of a catalyst or other non-participating agent.

[0056] In many embodiments, at least one of the crosslinkable entities comprises a polymer moiety linked by a crosslinking moiety. In some embodiments, at least one of the crosslinkable entities does not comprise a polymer moiety. In some embodiments, a pair of crosslinkable entities that react with each other (e.g., without administration of a catalyst or other non-participating agent) each comprise a polymer moiety linked by a crosslinking moiety. In some embodiments, a pair of crosslinkable entities that react with each other (e.g., without administration of a catalyst or other non-participating agent) comprises a first crosslinkable entity that comprises a polymer moiety linked by a crosslinking moiety and a second crosslinkable entity that does not comprise a polymer moiety.

[0057] In some embodiments, the crosslinkable entity comprises a polymeric portion and multiple crosslinking portions, which may be the same or different.In particular, the present disclosure provides insights and techniques relevant to achieving penetration of the crosslinkable entity into target sites in the skin (e.g., above, in, in, or below the epidermis, dermis, or underlying subcutaneous tissue).For example, in some embodiments, the present disclosure teaches that lipophilicity within a certain range can enable desirable (e.g., enhanced) penetration of the crosslinkable entity, particularly the crosslinkable entity that comprises a polymeric portion and a crosslinkable portion.

[0058] As shown in Figure 1, human skin is multi-layered, including the outer epidermis (itself a layered structure including the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale), the dermis, and the underlying hypodermis. The skin acts as a barrier to separate and protect the body from its environment. A major challenge in the cosmetic and dermatological fields is the development of techniques to enhance the penetration of compounds or drugs of interest across the skin. See, for example, Ng (2015) Skin Deep: The Basics of Human Skin Structure and DrugPenetration. In: Dragicevic N., Maibach H. (eds)Percutaneous PenetrationEnhancers Chemical Methods in Penetration Enhancement. Springer, Berlin,Heidelberg.

[0059] It is understood in the art that human skin limits the transport of compounds having molecular weights (e.g., number average molecular weights) greater than about 500 daltons (Bos and Meinardi, Experimental Dermatology, 9:165, 2000), and even for such small compounds, effective transport often requires additional manipulations, such as the administration of chemical or physical abrasive or disruptive agents, and / or electric currents or magnetic fields, etc.

[0060] The present disclosure provides, among other things, techniques relating to penetration across or through skin layers, and in particular such penetration by crosslinkable entities that react to produce crosslinked materials present at the target site. In many embodiments, the target site may be on, in, within or below the epidermis, dermis or underlying subcutaneous tissue. In particular, the present disclosure provides teachings that crosslinkable entities with improved skin penetration characteristics may be designed and / or prepared by modulating lipophilicity. For example, in some embodiments, the present disclosure teaches that the rate and / or extent of skin penetration by certain agents (in particular agents that are or include polymeric moieties and / or otherwise have number molecular weights (e.g., number average molecular weights) greater than 500 Daltons, and even in the range of 10-500 kD) may be enhanced by increasing the lipophilicity of the agent, for example by attaching one or more hydrophobic moieties to the agent. Those skilled in the art will recognize that in some embodiments, hydrophobic moieties that increase lipophilicity may be or include cross-linking moieties, and alternatively or additionally, in some embodiments, such hydrophobic moieties may not necessarily be or include cross-linking moieties and / or may not necessarily be involved in cross-linking (e.g., being present in and / or helping to produce a cross-linked material as described herein).

[0061] Alternatively or additionally, in some embodiments, the present disclosure provides techniques related to providing crosslinked materials to a target site in or on tissue (particularly in or on skin) such that contact between the material and the target site surface is maximized. In some embodiments, such surfaces may include one or more cavities or irregularities, which in some embodiments may be microscale or even nanoscale structures. In some embodiments, the present disclosure provides preparations of crosslinkable moieties with flow characteristics that promote such contact.

[0062] In some particular embodiments, crosslinkable entities for use according to the present disclosure comprise polymeric moieties linked by crosslinking moieties that impart increased lipophilicity to the conjugate (i.e., the crosslinkable entity) compared to the polymeric moieties alone, and as described herein, in some embodiments, such crosslinking moieties may also improve skin penetration by the crosslinkable entity compared to the skin penetration of the polymeric moieties alone.

[0063] In some embodiments, the penetration depth of the crosslinkable entity is assessed and / or described in terms of absolute distance (e.g., microns) below the skin surface. In some embodiments, the penetration depth of the crosslinkable entity is assessed and / or described in terms of number of cells below the skin surface.

[0064] In some embodiments, a crosslinkable entity as described herein can and / or does penetrate (e.g., when administered as described herein) to a particular depth into the skin, for example, within a certain period of time. In some embodiments, such particular depth may be, for example, at least 50 microns, at least 100 microns, at least 200 microns or more; alternatively or additionally, in some embodiments, such particular depth may be at least 2 cell layers, at least 3 cell layers, at least 4 cell layers, at least 5 cell layers, at least 6 cell layers, at least 7 cell layers, at least 8 cell layers, at least 9 cell layers, at least 10 cell layers or more, and / or such period of time may be, for example, within 1 day, 18 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour or less.

[0065] In some embodiments, at least 1% of a crosslinkable entity administered to the skin surface penetrates to a target site on, in, within, or below the epidermis, dermis, or underlying subcutaneous tissue, for example, within a period of one day. In some embodiments, the skin penetration characteristics of a crosslinkable entity as described herein are observed without any chemical or physical abrasive or disruptive agents, and / or electrical currents or magnetic fields, etc. (e.g., without penetration enhancers as understood in the art).

[0066] In some embodiments, the crosslinkable entity is characterized by a lipophilicity (logP) in the range of about -4 to about 2. In some embodiments, the crosslinkable entity described herein comprises a crosslinkable moiety that is lipophilic. In some embodiments, the lipophilicity of the crosslinkable moiety can be determined independently of the lipophilicity of the polymeric portion and / or the lipophilicity of the crosslinkable entity that comprises a polymeric portion and one or more crosslinkable moieties. In some embodiments, the crosslinkable moiety is characterized by a lipophilicity (logP) in the range of about 0 to about 6. For example, in some embodiments, the lipophilicity of the crosslinkable moiety is tested by inspection of the crosslinkable moiety prior to association and / or linkage with the molecular polymeric portion to form the crosslinkable entity.

[0067] In some embodiments, lipophilicity for a particular entity or moiety is determined by its partition coefficient (P) compared to a standard solvent (eg, octanol) and water or solutions thereof.

number

[0068] In some embodiments, the logP of an entity or moiety useful according to the present disclosure (eg, of a crosslinkable entity and / or of a crosslinking or other hydrophobic moiety) is greater than zero.

[0069] In some embodiments, the crosslinkable entity (e.g., a crosslinkable entity having lipophilic properties as described herein) has a molecular weight (e.g., number average molecular weight) of greater than 500 Daltons. In some embodiments, the crosslinkable entity has a molecular weight (e.g., number average molecular weight) in the range of 1-1000 kDa. In some embodiments, the system includes a crosslinkable entity that is about 10-250 kDa. In some embodiments, the system includes a crosslinkable entity that has a molecular weight (e.g., number average molecular weight) in the range of about 10-150 kDa. In some embodiments, the system includes a crosslinkable entity that has a molecular weight (e.g., number average molecular weight) in the range of about 10-100 kDa. In some embodiments, the system includes a crosslinkable entity that has a molecular weight (e.g., number average molecular weight) in the range of about 10-40 kDa. In some embodiments, the system includes a crosslinkable entity that has a molecular weight (e.g., number average molecular weight) of about 10 kDa. In some embodiments, the system includes a crosslinkable entity that has a molecular weight (e.g., number average molecular weight) of about 20 kDa. In some embodiments, the system includes a crosslinkable entity having a molecular weight (e.g., number average molecular weight) of about 30 kDa. In some embodiments, the system includes a crosslinkable entity having a molecular weight (e.g., number average molecular weight) of about 40 kDa. In some embodiments, the system includes a crosslinkable entity having a molecular weight (e.g., number average molecular weight) of about 50 kDa. In some embodiments, the system includes a crosslinkable entity having a molecular weight (e.g., number average molecular weight) of less than 10 kDa. In some embodiments, the system includes a crosslinkable entity having a molecular weight (e.g., number average molecular weight) of less than 100 kDa. In some embodiments, the system includes a crosslinkable entity having a molecular weight (e.g., number average molecular weight) of less than 250 kDa. In some embodiments, the system includes a crosslinkable entity having a molecular weight (e.g., number average molecular weight) of less than 500 kDa.

[0070] In some embodiments of the crosslinkable entity that comprises a polymer portion and a crosslinking portion, the crosslinking portion confers lipophilicity to the crosslinkable entity. In some embodiments, the identity and / or number and / or density of the crosslinking portion linked to a particular polymer portion in the crosslinkable entity are selected so that the crosslinkable portion has the characteristics as described herein. For example, in some embodiments, if a polymer portion is particularly hydrophilic, particularly long, and / or characterized by low skin penetration, particularly in its unlinked state, then a more lipophilic crosslinking portion (and / or a greater number / higher density of lipophilic crosslinking portions) is linked to that polymer portion.

[0071] Without wishing to be bound by any particular theory, the inventors propose that in some embodiments, it may be desirable to select a particular combination of polymer moieties and crosslinking moieties (and / or their number and / or density) to achieve three-dimensional packing of the crosslinkable entities within a certain volume, for example, to facilitate transport through one or more skin structures or layers (e.g., to make the crosslinkable entities "slippery" enough to pass through). In some embodiments, intra- and / or intermolecular interactions can contribute to the three-dimensional packing of the crosslinkable entities. In some embodiments, the three-dimensional packing of certain crosslinkable entities as described herein occurs by self-assembly (e.g., without the need for the addition of additional agents), at least under certain environmental conditions (e.g., physiological conditions).

[0072] Typically, the crosslinkable entity is biocompatible. For example, in some embodiments, administration of a crosslinkable entity as described herein does not cause significant irritation and / or inflammation (e.g., at the site of administration and / or at the target site).

[0073] In some embodiments, the crosslinkable entity is utilized in accordance with the present invention in a form or preparation having a viscosity suitable for topical application, e.g., sufficient viscosity to allow the applied crosslinkable entity to remain in contact with the administration site for a long enough period of time to allow penetration and / or otherwise distribution to the target site, rather than simply dripping, and in some such embodiments, the viscosity is within a range that allows the applied crosslinkable entity to flow into microstructures on the skin surface. In some embodiments, the crosslinkable entity is utilized in accordance with the present invention in a form or preparation having a viscosity suitable for administration by injection. In some embodiments, a suitable viscosity is one that allows the crosslinkable entity to be administered topically and / or rubbed into the skin without running off rapidly.

[0074] In some embodiments, the present disclosure provides systems that include two or more crosslinkable entities that interact with each other to form a crosslinked material (e.g., to provide a crosslinked material in situ at a target site on, in, within or beneath the epidermis, dermis or underlying subcutaneous tissue).

[0075] In some embodiments, at least one crosslinkable entity of the provided system comprises a polymer moiety linked by a crosslinking moiety. In some such embodiments, at least one crosslinkable entity of the system does not comprise a polymer moiety linked by a crosslinking moiety (i.e., comprises or consists of a crosslinking moiety but does not comprise a polymer moiety), and in some such embodiments, only one crosslinkable entity of the provided system comprises a polymer moiety linked by a crosslinking moiety. Alternatively, in some embodiments, each crosslinkable entity of the provided system comprises a polymer moiety linked by a crosslinking moiety, and in some such embodiments, each crosslinkable entity of the provided system comprises the same polymer moiety.

[0076] The different crosslinkable entities of the provided systems contain complementary crosslinking moieties such that the crosslinkable entities react to form a crosslinked material, and in some embodiments, such crosslinking occurs without the addition of any catalyst.

[0077] In some embodiments, at least one crosslinkable entity of a provided system has a molecular weight (e.g., number average molecular weight) greater than 500 Daltons as described herein (e.g., has a molecular weight (e.g., number average molecular weight) in the range of 1-1000 kDa). In some embodiments, at least one crosslinkable entity of a provided system has a molecular weight (e.g., number average molecular weight) in the range of 1-10,000 kDa as described herein. In some such embodiments, at least one crosslinkable entity of a system does not have a molecular weight (e.g., number average molecular weight) greater than 500 Daltons as described herein (e.g., in the range of 1-400 Daltons), and in some such embodiments, only one crosslinkable entity of a provided system has such a high molecular weight (e.g., number average molecular weight). Alternatively, in some embodiments, each crosslinkable entity of a provided system has such a high molecular weight (e.g., number average molecular weight).

[0078] Polymer Part In some embodiments, the invention encompasses crosslinkable entities that include a polymer portion and a crosslinking portion. In some embodiments, one or more of the polymer portions are glycosaminoglycans or polysaccharides. In some embodiments, "polysaccharides" include dextran, starch, or pectin. In some embodiments, one or more of the polymer portions are dextran. In some embodiments, one or more of the polymer portions are starch. In some embodiments, one or more of the polymer portions are pectin. In some embodiments, "glycosaminoglycans" include hyaluronic acid (HA), heparin sulfate, chondroitin sulfate, dermatan sulfate, and keratin sulfate. In some embodiments, the polymer is hyaluronic acid.

[0079] In some embodiments, one or more of the polymer moieties are synthetic polymers. In some embodiments, "synthetic polymer" includes PEG, PEG-diamine, polyacrylic acid, N-(2-hydroxypropyl) methacrylamide (HPMA), polycaprolactone (PCL) or poly(lactic-co-glycolic acid) (PLGA). In some embodiments, the synthetic polymer is PEG. In some embodiments, the synthetic polymer is PEG-diamine. In some embodiments, the synthetic polymer is polyacrylic acid. In some embodiments, the synthetic polymer is HPMA. In some embodiments, the synthetic polymer is PCL. In some embodiments, the synthetic polymer is PLGA.

[0080] In some embodiments, one or more of the polymer moieties is a polypeptide (e.g., a protein). In some embodiments, the useful polypeptide is or comprises collagen, gelatin, elastin, or a functional fragment thereof. In some embodiments, the polypeptide is or comprises collagen or a functional fragment thereof. In some embodiments, the polypeptide is gelatin. In some embodiments, the polypeptide is elastin.

[0081] In some embodiments, one or more polymer moieties included in the system as described herein are or include HA. In some embodiments, one or more polymer moieties included in the system as described herein are or include glycosaminoglycans. In some embodiments, one or more polymer moieties included in the system as described herein are or include polypeptides (e.g., non-natural polypeptides and / or synthetic or recombinant polypeptides). In some embodiments, the provided system includes a plurality of different crosslinkable entities that include polymer moieties, and in some such embodiments, the polymer moieties are selected from the group consisting of HA, proteoglycans, polypeptides, and combinations thereof.

[0082] bridging part As described herein, the present disclosure provides a system comprising two or more crosslinkable entities that are compatible with each other in that they react to form an in situ crosslinked material.

[0083] Those skilled in the art are aware of a variety of chemical cross-linking systems. In many embodiments, the present disclosure utilizes cross-linking chemistries that do not require the addition of a catalyst.

[0084] In some embodiments, one or more of the crosslinkable entities of the system comprises a crosslinking moiety. In some embodiments, a "crosslinking moiety" is capable of participating in a Click reaction.

[0085] One of skill in the art will recognize that the Click reaction may be a two or more part reaction that brings together two or more substrates and occurs under physiological pH. In some embodiments, the Click reaction exhibits suitable kinetics (e.g., a second order rate constant (k2) of about 9M -1 s -1 (It is.)

[0086] For example, in some embodiments, the crosslinking chemistries may include, but are not limited to, cycloaddition, nucleophilic substitution reactions, condensation reactions, and nucleophilic addition reactions. In some embodiments, the click reaction is a [3+2] cycloaddition, a [4+2] cycloaddition, or a [4+1] cycloaddition. In some embodiments, the click reaction is an azide-alkyne cycloaddition, a nitrone-olefin cycloaddition, or a Diels-Alder reaction. In some embodiments, the click reaction is a Schiff reaction, a Michael-type addition, a nucleophilic substitution reaction on a haloacetate, formation of a disulfide linkage, free radical polymerization, a Huysgen reaction, an enzyme reaction to catechol, or a cycloaddition reaction. In some embodiments, the click reaction is a reaction between D-cysteine ​​(CYS) and phenol (tyramine), which spontaneously crosslinks after selective oxidation, or cyanobenzothiazole (CBT). In some embodiments, the click reaction is a reaction between CBT and D-cysteine. In some embodiments, the click reaction is a reaction between CBT and L-cysteine. In some embodiments, the CBT may include an analog of CBT, e.g., isotopically labeled CBT. In some embodiments, the CBT includes a substitution analog of CBT. In some embodiments, the CYS may include an analog of CYS, e.g., isotopically labeled CYS. In some embodiments, the CYS includes a substitution analog of CYS.

[0087] In some embodiments, the present disclosure recognizes that certain cross-linking moieties may be particularly useful in in situ cross-linking situations as described herein.

[0088] For example, in some embodiments, the present disclosure utilizes one or more bridging moieties that are characterized by a desired degree of lipophilicity when linked to, for example, a particular polymer moiety. By way of example only, CBT is representative of a crosslinkable moiety that can be linked to a polymer moiety in a crosslinkable entity useful as described herein. Those skilled in the art who read the present disclosure will recognize that in some embodiments, the lipophilicity of a crosslinkable entity that includes a particular polymer moiety can be adjusted through the linkage of multiple hydrophobic moieties (e.g., hydrophobic bridging moieties) that can be the same or different (not all of which need to be crosslinking moieties).

[0089] Further, in some embodiments, the cross-linking moiety is or comprises an azide, alkyne, nitrone, olefin, diene, tetrazine, isocyanate, Michael acceptor, enone, aldehyde, amine, α-halocarbonyl moiety, maleimide, thiol, CBT, D-cysteine, acrylic residue, phenol, tyramine, or catechol. In some embodiments, the cross-linking moiety is or comprises D-cysteine. In some embodiments, the cross-linking moiety is or comprises L-cysteine. In some embodiments, the reactive moiety is or comprises CBT.

[0090] In some embodiments, at least one cross-linking moiety included within the provided systems can be utilized without linkage to a polymer moiety, i.e., in some embodiments, at least one cross-linkable entity included within the provided systems can consist of a cross-linking moiety or can include a cross-linking moiety and at least one other moiety that is not a polymer moiety (which other moiety may be or can include a hydrophobic moiety in some embodiments, as will become apparent from this disclosure).

[0091] In some embodiments, the provided system may utilize a cross-linking moiety that is or includes, for example, a diamine, a peptide, a dithiol, or a dihydrazide. In some such embodiments, the diamine may be ethylene diamine, such as polyethylene glycol (PEG) diamine, and in some such embodiments, the dihydrazide may be oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, or pimelic acid dihydrazide. Without being bound by semantics, it is recognized that certain such compounds may be described as consisting of a cross-linking moiety or as comprising one or more cross-linking moieties (e.g., each of which may be a single reactive atom or a small number of atoms) and a polymeric portion that may include only a very small number of "monomers" (e.g., two or three) (in some cases, each such "monomer" may be composed of only a small number of atoms). Nonetheless, those of skill in the art reading this disclosure will recognize that these compounds may, in various embodiments, be utilized in conjunction with other cross-linking entities having complementary cross-linking moieties as described herein.

[0092] Other parts In some embodiments, a crosslinkable entity as described herein can include one or more moieties other than the polymeric or crosslinking moieties, which in some embodiments can be covalently associated with the polymeric and / or crosslinking moieties. In some embodiments, the "other" moieties can be releasably associated with the crosslinkable entity (e.g., via a cleavable bond; in some such embodiments, such cleavable bond can be cleaved at the target site).

[0093] For example, in some embodiments, the crosslinkable entity may include a drug moiety (which in some embodiments may itself be in a prodrug form).

[0094] In some embodiments, the drug moiety is or includes a synthetic or natural small molecule or biomolecule (eg, a carbohydrate, lipid, nucleic acid, polypeptide or analog, or a combination thereof).

[0095] In some embodiments, the drug moiety may be a drug moiety that improves the appearance of the skin in one or more ways. For example, in some such embodiments, the drug moiety may brighten the skin, clear breakouts, firm, improve cellular activity in the skin, improve collagen synthesis, improve wound healing profile, improve pigmentation, improve skin barrier function, normalize healthy microflora via topical prebiotics, It can prevent damage (e.g., due to UV exposure), reduce the appearance of scars, reduce inflammation, reduce itching, reduce redness, heal wounds, soothe, or treat burns, or a combination thereof.

[0096] In some embodiments, the drug moiety can include an alpha-hydroxy acid (e.g., lactic acid, tartaric acid, or citric acid), an antioxidant (e.g., glutathione, isoflavones, polyphenols (e.g., resveratrol), or selenium), a beta-hydroxy acid (e.g., salicylic acid), a polyhydroxyl acid (e.g., gluconolactone or lactobionic acid), hydroquinone, a natural skin lightening agent (e.g., kojic acid), a retinoid (e.g., retinoic acid, retinol, tretinoin or derivatives thereof), a ceramide, a peptide, an amino acid, a curcuminoid, a vitamin (and its derivatives) (e.g., L-ascorbic acid, vitamin B, niacinimide, and vitamin K), a sunscreen (e.g., oxybenzone, avobenzone, octisalate, octocrylene, homosalate, octinoxate, meroxyl (SX and XL), or a metal oxide), a colorant, a pigment, or a natural botanical, or a combination or prodrug thereof.

[0097] In some embodiments, the drug moiety can include an anti-inflammatory agent (e.g., a corticosteroid, a nonsteroidal anti-inflammatory agent (crisaborole)), an antibiotic (e.g., clindamycin or ketoconazole), an antifungal agent (e.g., clotrimazole or ketoconazole), an anti-acne agent (e.g., a retinoid or salicylic acid), an analgesic, an anti-cancer agent or an anti-proliferative agent, an agent that treats erythema (e.g., oxymetazoline hydrochloride), an agent that reduces subcutaneous fat (e.g., deoxycholate), a hair growth agent (e.g., finasteride), or a combination or prodrug thereof.

[0098] target site The techniques as described herein are useful for providing a crosslinked material in situ at a target site. In some embodiments, the target site is or includes (e.g., on or in) a body tissue. Described herein are techniques for applying a material to an application site such that a crosslinked material is provided (e.g., formed) at the target site.

[0099] For example, in some embodiments, the body tissue is or comprises epithelial tissue. In some embodiments, the body tissue is or comprises connective tissue. In some embodiments, the body tissue is or comprises nervous tissue. In some embodiments, the body tissue is or comprises muscle tissue. In some embodiments, the body tissue is or comprises eye tissue, skin tissue, or subcutaneous tissue. In some embodiments, the body tissue is or comprises subcutaneous fat, corneal epithelium, or mucosa.

[0100] As described herein, in many embodiments, the material (i.e., one or more crosslinkable entities) is administered to the application site such that the crosslinkable material is provided in situ at the target site (e.g., formed at the target site). In many embodiments, the target site is the site reached after application to a surface, e.g., a tissue surface. In some embodiments, the tissue surface is a surface of a tissue (e.g., skin, eye, or certain mucous membranes) exposed on the surface of an organism. In some embodiments, the tissue surface is a surface of an internal tissue that can be accessed or exposed by performing, for example, a procedure (e.g., a medical procedure, e.g., a surgical procedure, including an orthoscopic procedure) or process applied to an organism.

[0101] In some embodiments, the target site is a target site on, in, in, or below the stratum corneum. In some embodiments, the target site is a site on, in, in, or below the epidermis. In some embodiments, the target site is a target site on, in, in, or below the dermis.

[0102] In some embodiments, the target site can be selected to be or include damaged skin (e.g., due to a disease, disorder or condition, or surgical, cosmetic or dermatological treatment). In some embodiments, damaged skin can be or include skin that has been treated with a laser (e.g., fractional laser), intense-pulsed-light treated skin, microneedle treated skin, skin that has been treated with a chemical peel, skin that has been exposed to skin abrasion, skin that has been damaged by trauma (including by surgery), skin that is affected by a medical condition, such as atrophic skin, extreme dryness, etc.

[0103] Alternatively or additionally, in some embodiments, the target site may be selected to treat skin that has wrinkles (e.g., crow's feet or other facial wrinkles) or that otherwise shows signs of aging, or to improve the appearance of additional plumpness. ) and / or skin where resilience is desired.

[0104] In certain embodiments, the target skin may be or include skin in an area selected from the group consisting of both lips, the lower lip, the upper lip, dark circles under the eyes or other areas around the eyes (e.g., crow's feet), nasolabial folds, the forehead, the cheeks, the backs of the hands, the earlobes, the knees, the neck, the décolleté, the arms, the legs, the torso, the buttocks, the feet, and combinations thereof, including, for example, the entire face.

[0105] In some embodiments, the target site may be or include adipose tissue (e.g., subcutaneous adipose tissue). Alternatively or additionally, in some embodiments, the target site may be or include a gland, such as a sebaceous gland and / or a serous gland (e.g., sweat gland), and / or the target site may be or include a vessel (e.g., blood vessel) or duct (e.g., sweat duct). Still further alternatively or additionally, in some embodiments, the target site may be or include one or more specific cell populations that may be localized, for example, within one or more skin layers, for example, in some such embodiments, the relevant cell population may be or include a particular stem cell or cell population associated with a disease or injury condition. In some particular embodiments, the target site may be or include a pigment-producing cell. In some particular embodiments, the target site may be or include one or more hair follicles. In some particular embodiments, the target site may be or include one or more neuronal cells or nerves.

[0106] Administration of the provided system As described herein, the present disclosure provides a system for administration to a skin site that includes at least a first crosslinkable entity and a second crosslinkable entity. In some embodiments, both crosslinkable entities are administered to the same site, and in some embodiments, each can be administered to a different site, so long as both ultimately reach the intended target site, and thus the crosslinked material produced by the reaction of the first crosslinkable entity and the second crosslinkable entity is formed or otherwise provided in situ at the target site.

[0107] In some embodiments, the at least one crosslinkable entity is administered topically (i.e., to the skin surface). In some embodiments, the at least one crosslinkable entity is administered parenterally, for example, by injection at the target site or at a location from which the entity penetrates to the target site.

[0108] In some embodiments, a provided system is administered to the face of a subject (e.g., the entire face and / or specific targets on the face of a subject, such as the lips, lower lip, upper lip, dark circles under the eyes, crow's feet, nasolabial folds, forehead, cheeks, or combinations thereof). In some embodiments, a provided system is administered to a site other than the face (e.g., the knee, neck, décolleté, leg, arm, torso, buttocks, or foot). In some embodiments, a provided system is administered to the hand (e.g., the back of the hand). In some embodiments, a provided system is administered to the earlobe.

[0109] In some embodiments, the first crosslinkable entity and the second crosslinkable entity are administered simultaneously.For example, in some embodiments, the first crosslinkable entity and the second crosslinkable entity can be combined before or when they are administered.In some embodiments, such combination can be called a premix and can be prepared a certain period before administration, typically such a premix is ​​prepared within about 1 minute before administration.

[0110] In some embodiments, the administration site is prepared prior to administration of the crosslinkable entity. In some embodiments, the administration site is prepared by washing the site with warm water and soap. In some embodiments, the administration site is prepared by tape stripping. In some embodiments, tape stripping comprises applying translucent Scotch tape to the administration site. In some embodiments, tape stripping further comprises removing previously applied translucent Scotch tape from the administration site. In some embodiments, tape stripping is repeated until the administration site is shiny. In some embodiments, tape stripping is repeated at least 40 times.

[0111] In some embodiments, the first crosslinkable entity and the second crosslinkable entity are administered sequentially, and in some such embodiments, a period of time separates the administration of the first crosslinkable entity and the second crosslinkable entity. In some embodiments, such a period of time is about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 6 hours, about 12 hours, or longer. In some embodiments, such a period of time is about 2 hours. In some embodiments, such a period of time is less than about 12 hours, less than about 6 hours, less than about 1 hour, less than about 20 minutes, less than about 10 minutes, less than about 5 minutes, less than about 2 minutes, or less than about 1 minute.

[0112] In some embodiments, the application site is covered after application of the crosslinkable entity. In some embodiments, the application site is covered with a Tegaderm™ type film after application of the crosslinkable entity.

[0113] In some embodiments, the period between administration of the first crosslinkable entity and the second crosslinkable entity is sufficient to allow substantial penetration of the first crosslinkable entity before the second crosslinkable entity is administered. In some embodiments, the period is sufficient to allow at least about 1%, at least about 2%, at least about 5%, at least about 10%, or at least about 20% of the first crosslinkable entity to penetrate before the second crosslinkable entity is administered. In some embodiments, the period is sufficient to allow the first agent to not degrade and / or be removed from the skin. The kinetics and / or extent of penetration for a particular crosslinkable entity can be determined, for example, as described herein, including by using a model system (e.g., pig skin), and the relevant period can be selected in light of such determination.

[0114] In some embodiments, the skin is treated with water after administration of the system.

[0115] In some embodiments, the system is administered daily. In some embodiments, the system is administered at least once a day. In some embodiments, the system is administered at least twice a day. In some embodiments, the system is administered 1-5 times a day. In some embodiments, the system is administered 3-5 times a day. In some embodiments, the system is administered every 3 days. In some embodiments, the system is administered every 7 days. In some embodiments, the system is administered about every 15 days. In some embodiments, the system is administered about every 30 days. In some embodiments, the system is administered about every 60 days. In some embodiments, the system is administered about every 90 days.

[0116] In some embodiments, the provided systems include a chemical entity that improves skin penetration for the crosslinkable entity. In some embodiments, the chemical entity is administered simultaneously with the crosslinkable entity. In some embodiments, the chemical entity and the crosslinkable entity are administered at different times.

[0117] In some embodiments, the provided systems include one or both of a permeation inhibitor (e.g., which may interact with the crosslinkable entity and / or otherwise retard permeation) and a crosslinking inhibitor (e.g., which may block one or more features of the crosslinkable entity or crosslinking moiety or otherwise interfere with reaction of the crosslinking moiety to produce crosslinks), and in some such embodiments, such inhibitors can be removed (e.g., by diffusion, washing, degradation) before, during, during or after administration of the crosslinkable entity.

[0118] In some embodiments, one or more crosslinkable entities are administered as or in a sustained release formulation. In some embodiments, one or more crosslinkable entities are encapsulated within a matrix or particle (e.g., nanoparticle). In some embodiments, one or more crosslinkable entities are provided as or in an emulsion or dispersion.

[0119] In some embodiments, the crosslinkable entities, whose crosslinking moieties interact to form crosslinks in the crosslinked material, are administered in relative amounts (e.g., formulated for administration in relative amounts) such that the complementary crosslinking moieties are present in stoichiometric amounts. Alternatively, in some embodiments, such crosslinkable entities are administered in relative amounts such that one of a pair of complementary crosslinking moieties is present in molar excess relative to the other, and in some such embodiments, such molar excess is in the range of 1.1:1 to 10,000:1. In some embodiments, the molar excess of the relative amounts of the crosslinkable entities is in the range of 1.1:1 to 2:1. In some embodiments, the molar excess of the relative amounts of the crosslinkable entities is in the range of 1.1:1 to 10:1. In some embodiments, the molar excess of the relative amounts of the crosslinkable entities is in the range of 1.1:1 to 100:1. In some embodiments, the molar excess of the relative amounts of the crosslinkable entities is in the range of 1.1:1 to 1,000:1.

[0120] In some embodiments, the formulation comprises from about 0.001% w / w to about 5.00% w / w of the crosslinkable entity. In some embodiments, the formulation comprises from about 0.01% w / w to about 5.00% w / w of the crosslinkable entity. In some embodiments, the formulation comprises from about 0.1% w / w to about 5.00% w / w of the crosslinkable entity. In some embodiments, the formulation comprises from about 1% w / w to about 5.00% w / w of the crosslinkable entity. In some embodiments, the formulation comprises from about 1% w / w to about 3% w / w of the crosslinkable entity. In some embodiments, the formulation comprises about 2% w / w of the crosslinkable entity. In some embodiments, the formulation comprises PBS and about 2% w / w of the crosslinkable entity.

[0121] Local application In some embodiments, at least one crosslinkable entity of the provided system is provided topically, for example, at a site on the skin, for example, on the surface of the skin.

[0122] In some embodiments, the formulation for topical administration may be or may include a cream, gel, liquid, lotion, mask, matrix, mist particles, paste, patch, powder, serum, solid, spray (or collection thereof), or combinations thereof. In some embodiments, the formulation for topical administration may be or may include a mask or serum.

[0123] In some embodiments, formulations for topical administration can include antioxidants, buffering agents, chelating agents, clarity enhancers, emollients, emulsifiers, gelling agents, humectants, lubricants, moisturizers, pH adjusters, preservatives, protectants, prebiotics, probiotics, rheology modifiers, sensory modifiers, soothing agents (e.g., soothing botanical serums), stabilizers, sunscreens (e.g., broad spectrum inorganic sunscreens), or thickeners.

[0124] In some embodiments, topical application does not include administration or application of a penetration enhancer (eg, a chemical or physical abrasive or disruptive agent, and / or an electrical current, and / or an electric or magnetic field, etc.).

[0125] In some embodiments, the first crosslinkable entity and the second crosslinkable entity are separated by chemical means, hi some such embodiments, the crosslinkable entities are encapsulated, for example, in micelles, liposomes, etc.

[0126] In some embodiments, the system is administered and the crosslinkable entity is released over a period of time.

[0127] In some embodiments, the system comprises a transdermal patch formulated to promote sustained release of the crosslinkable entity. Those skilled in the art will recognize that the transdermal patch may be a single-layer drug-type patch, a multi-layer drug-type patch, a reservoir, or a matrix. As discussed below, in some embodiments, the patch may comprise microneedles (some or all of which may be solid protrusions in some embodiments, as known in the art, some or all of which may be hollow in some embodiments, as known in the art).

[0128] Those of skill in the art reading this disclosure will recognize that in some embodiments, delivery can be achieved or enhanced by local administration to an area that is or has been damaged by exposure to, for example, one or more of laser (e.g., fractional laser) treatment, intense-pulsed-light treatment, microneedle treatment, chemical peel treatment, skin abrasion, etc., each of which treatments may, in some embodiments, be administered or may have been administered before, during, or after topical administration of a system as described herein.

[0129] injection In some embodiments, at least one crosslinkable entity is administered by injection. In some embodiments, the injection is below the skin surface. In some embodiments, the injection is into a target site. In some embodiments, the injection is into a site from which the injected crosslinkable entity penetrates into the target site.

[0130] In some embodiments, injection can include the application of force or pressure (eg, with a syringe) such that delivery is accomplished, for example, through a needle, cannula, or other passageway.

[0131] In some embodiments, the injection can be performed through a microneedle (e.g., may be associated with a patch). For example, in some embodiments, one or more crosslinkable entities, or combinations thereof, can be administered through a microneedle (e.g., as a liquid); alternatively or additionally, in some embodiments, one or more crosslinkable entities, or combinations thereof, can be disposed within one or more microneedles that can dissolve or degrade upon application or thereafter. Those skilled in the art will recognize that the use of dissolving or degrading microneedles may be particularly useful or desirable for delivering relatively viscous materials (e.g., partially or fully formed crosslinked materials as described herein). In some embodiments, one or more crosslinkable entities may be disposed within the microneedle, e.g., within its lumen (e.g., perforations), and in some such embodiments, one or more crosslinkable entities (or crosslinked materials formed therefrom) can be released by injection through the lumen and / or by dissolution or degradation of the microneedle (e.g., its wall). In some embodiments, the crosslinkable entity or entities may be integrally formed with the microneedle, or in some such embodiments, the microneedle may be formed from a material that dissolves or degrades upon or after application, thereby releasing the crosslinkable entity or entities, or crosslinked material formed therefrom.

[0132] In some embodiments, the injection is performed with a dual bore syringe or needle. In some such embodiments, the first crosslinkable entity and the second crosslinkable entity are maintained in separate compartments of the syringe or needle, at least until administration. In some embodiments, the first crosslinkable entity and the second crosslinkable entity are combined during administration, while in other embodiments, they are maintained separately during administration (i.e., each is administered separately, optionally at times separated by a period of time, as described herein).

[0133] In some embodiments, the injection is performed with a needle having a bore size of about 26 gauge to about 30 gauge.

[0134] In some embodiments, the skin is pretreated prior to administration of the system (e.g., immediately or immediately prior to administration). In some embodiments, pretreatment of the skin is achieved by administering or applying a permeabilizing agent or device. In some embodiments, pretreatment of the skin includes one or more of the following: application of an abrasive cleanser or chemical peel, skin abrasion, electroporation, iontophoresis, low frequency sonophoresis, microneedle treatment. In some embodiments, the skin is abraded prior to administration of the system. In some such embodiments, the skin is abraded with microneedles and / or fractional laser prior to administration of the system. In some embodiments, the crosslinkable entity is administered after pretreatment of the skin with microneedles (e.g., immediately or immediately after pretreatment).

[0135] kit In some embodiments, the present disclosure provides a kit comprising a system described herein.

[0136] In some embodiments, provided kits include multiple containers or vessels, including containers that separately house a first crosslinkable entity and a second crosslinkable entity.

[0137] In some embodiments, the kit provided includes at least one container or vessel containing multiple separate compartments (e.g., a dual-perforated syringe or needle, or a dual-chamber package having a pre-dispensing mixing chamber), and in some embodiments, the first crosslinkable entity and the second crosslinkable entity are contained separately in such compartments.

[0138] In some embodiments, the present disclosure provides one or more containers, vessels, or compartments in which a first or second crosslinkable entity as described herein is disposed. In some such embodiments, the disposed crosslinkable entity is present in dry form, and in some such embodiments, the disposed crosslinkable entity is present in liquid form. In some such embodiments, the disposed crosslinkable entity is stored for a period of time (e.g., at least one day, one week, one month, three months, six months, or longer), and in some such embodiments, the stored composition is stabilized over a storage period during which at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the crosslinkable entity remains undegraded. In some embodiments, the stored composition is stabilized over a storage period during which at least about 50%, 65%, 70%, 75% or more of the crosslinkable entity remains undegraded.

[0139] Characterization Those of skill in the art reading this specification will recognize that it may be desirable to characterize one or more features of the crosslinkable entities, and / or of their components or combinations, and / or of the crosslinked materials they produce, for example, when designing (e.g., selecting appropriate components thereof) or producing a provided system, and / or when monitoring or assessing its preparation. Alternatively or additionally, in some embodiments it may be desirable to assess one or more features of a provided system when administered, for example, to a subject or to monitor its treatment.

[0140] In some embodiments, the first and second crosslinkable entities of the system react with each other to form a crosslinked material when combined in vitro under physiological conditions at 37° C. in a period of less than about 60 minutes. The properties of such crosslinked materials can be modulated by the selection of the crosslinkable entities that produce the crosslinked material and can be characteristic traits of the particular crosslinked material provided by the present invention. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 1 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 500 Pa. In some embodiments, the storage modulus of the crosslinked material is in the range of 50 Pa to 100 Pa. In some embodiments, the storage modulus of the crosslinked material is in the range of 100 Pa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 500 Pa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 1 kPa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 5 kPa to 10 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 500 Pa to 5 kPa. In some embodiments, the storage modulus of the crosslinked material is in the range of 500 Pa to 1 kPa.

[0141] In some embodiments, the crosslinked material loses less than 20% mass due to degradation over 3 days in physiological buffer. In some embodiments, the crosslinked material loses less than 10% mass due to degradation over 3 days in physiological buffer. In some embodiments, the crosslinked material loses less than 5% mass due to degradation over 3 days in physiological buffer.

[0142] In some embodiments, the swelling degree of the crosslinked material upon hydration is a characteristic property of the crosslinked material. In some embodiments, the swelling degree of the crosslinked material is determined by the mass ratio of the dry crosslinked material to the fully swollen crosslinked material in equilibrium with an external aqueous buffer (Q=m swollen / m dry ) is measured based on the crosslinked dry material and the crosslinked fully swollen material. In some embodiments, the ratio of the crosslinked dry material to the crosslinked fully swollen material is in the range of 50 to 1000. In some embodiments, the ratio of the crosslinked dry material to the crosslinked fully swollen material is in the range of 100 to 1000. In some embodiments, the ratio of the crosslinked dry material to the crosslinked fully swollen material is in the range of 500 to 1000. In some embodiments, the ratio of the crosslinked dry material to the crosslinked fully swollen material is in the range of 750 to 1000. In some embodiments, the ratio of the crosslinked dry material to the crosslinked fully swollen material is in the range of 50 to 100. In some embodiments, the ratio of the crosslinked dry material to the crosslinked fully swollen material is in the range of 50 to 250. In some embodiments, the ratio of the crosslinked dry material to the crosslinked fully swollen material is in the range of 50 to 500. In some embodiments, the ratio of the crosslinked dry material to the crosslinked fully swollen material is in the range of 50 to 750. In some embodiments, the ratio of crosslinked dry material to crosslinked fully swollen material is in the range of 250 to 750. In some embodiments, the ratio of crosslinked dry material to crosslinked fully swollen material is in the range of 400 to 600.

[0143] In some embodiments, the time at which the formation of a crosslinked material occurs is determined by the time from when the first crosslinkable entity and the second crosslinkable entity of the system are combined until the dynamic storage modulus (G') becomes greater than the loss modulus (G''). In some embodiments, the first and second clickable entities form a crosslinked material in less than 1 second. In some embodiments, the first and second clickable entities form a crosslinked material in less than 10 seconds. In some embodiments, the first and second clickable entities form a crosslinked material in less than 1 minute. In some embodiments, the first and second clickable entities form a crosslinked material in less than 10 minutes. In some embodiments, the first and second clickable entities form a crosslinked material in less than 30 minutes. In some embodiments, the first and second clickable entities form a crosslinked material in less than 60 minutes.

[0144] In vitro characterization In some embodiments, characterization may include applying a provided system to a model skin system (e.g., pig skin).

[0145] In some embodiments, the characterization can include monitoring one or more features of skin penetration. In some embodiments, the amount of crosslinkable entity that penetrates the skin 0.5-2 days after topical application into pig skin is quantified, for example, after solubilizing the skin using enzymes and / or other solubilizing agents. In some embodiments, the amount of crosslinkable entity that penetrates the skin at least 0.5 days after topical application into pig skin is quantified after solubilizing the skin using enzymes and / or other solubilizing agents. In some embodiments, the amount of crosslinkable entity that penetrates the skin less than 2 days after topical application into pig skin is quantified after solubilizing the skin using enzymes and / or other solubilizing agents. In some embodiments, the amount of crosslinkable entity that penetrates after topical application into pig skin, comprising a polymeric portion and a crosslinking portion, exceeds the amount of an entity that comprises the same polymeric portion and does not comprise a crosslinking portion. In some embodiments, the crosslinkable entity comprises HA, and the amount of crosslinkable entity that penetrates after topical application into pig skin exceeds the amount of natural HA of the same molecular weight (e.g., number average molecular weight) that penetrates the skin. Preferably, in some embodiments, the amount of crosslinkable entity comprising HA that penetrates after topical application into pig skin exceeds the natural amount of HA already found in the skin (natural amount of HA already found in pig skin = approximately 100-800 ug per gram of dry tissue).

[0146] In some embodiments, the penetration depth of the crosslinked material in the porcine skin is measured by shining an ultraviolet light on the target site. In some embodiments, the presence of the crosslinked material in the porcine skin is determined by observing fluorescence upon shining an ultraviolet light on the target site.

[0147] In some embodiments, the presence of the crosslinked material at the target site becomes observable after a period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or more days). In some embodiments, the presence of the crosslinked material at the target site becomes observable after about 3.5 days. In some embodiments, the presence of the crosslinked material at the target site becomes observable after about 9 days.

[0148] In vivo characterization Upon administration of the system described herein, the contribution of the target site and / or administration site, i.e., the skin, is evaluated at an appropriate time for improvement compared to the skin characteristics before administration of the system. In some embodiments, the skin characteristics include wrinkles, brightness, firmness, water content, skin thickness, elasticity, and skin smoothness. In some embodiments, the improvement includes repair of atrophic skin or scars. In some embodiments, administration of the system results in enhanced fibroblast activation and / or collagen synthesis within the target site of the skin.

[0149] In some embodiments, skin improvement is measured by the Global Aesthetic Improvement Scale (GAIS). One skilled in the art will appreciate that the GAIS is based on the following scale: It will be appreciated that the distance may be determined using a constant scale. 1 = Significantly improved 2=Much improved 3=Improved 4 = No change 5=worsening

[0150] In some embodiments, the majority of subjects observe an improvement of >1 on the GAIS. In some embodiments, the majority of subjects observe an improvement of >2 on the GAIS. In some embodiments, the majority of subjects observe an improvement of >3 on the GAIS. In some embodiments, the majority of subjects observe an improvement of about 1 to about 2 on the GAIS. In some embodiments, the majority of subjects observe an improvement of about 1 to about 3 on the GAIS. In some embodiments, the majority of subjects observe an improvement of about 1 on the GAIS. In some embodiments, the majority of subjects observe an improvement of about 2 on the GAIS. In some embodiments, the majority of subjects observe an improvement of about 3 on the GAIS.

[0151] In some embodiments, skin improvement is measured using the Modified Fitzpatrick Wrinkle Scale (MFWS). Those skilled in the art will recognize that the MFWS can be determined using the following rating scale: Class 0 - No wrinkles. No visible wrinkles or continuous skin lines. Class 0.5 - Very shallow but visible wrinkles. Class 1 - Fine wrinkles. Visible wrinkles and slight dents. Class 1.5 - Visible wrinkles and obvious depressions. Wrinkle depth 0-1mm. Class 2 - Moderate wrinkles. Clearly visible wrinkles. Wrinkle depth 1-2mm. Class 2.5 - Pronounced and visible wrinkles. Wrinkle depth >2mm and <3mm. Class 3 - Deep wrinkles. Deep groove-like wrinkles. Wrinkle depth greater than 3mm.

[0152] In some embodiments, the subject's wrinkles are reduced by at least 0.5 MFWS. In some embodiments, the subject's wrinkles are reduced by between 1 and 0.5 MFWS. In some embodiments, the subject's wrinkles are reduced by between 1.5 and 0.5 MFWS. In some embodiments, the subject's wrinkles are reduced by between 2 and 0.5 MFWS. In some embodiments, the subject's wrinkles are reduced by between 2.5 and 0.5 MFWS. In some embodiments, the subject's wrinkles are reduced by between 3 and 0.5 MFWS.

[0153] In some embodiments, the subject's skin is assessed for improvement in smoothness, radiance, or firmness.

[0154] In some embodiments, the moisture content of skin can be monitored. In some embodiments, the moisture content of skin is higher than untreated skin as determined by methods known in the art. In some embodiments, the moisture content of skin is determined by Corneometer CM 825. In some embodiments, the moisture content of skin is >20% higher on the 7th day after administration of the system.

[0155] In some embodiments, the provided system is used to treat a human exhibiting abnormal transepidermal water loss (TEWL). One skilled in the art will appreciate that abnormal TEWL can be the result of skin damage caused, for example, by burns, certain chemicals, pathological conditions (e.g., eczema), physical abrasion, tape stripping, ultraviolet radiation, or a combination thereof. In some embodiments, the level of TEWL of treated skin is lower compared to untreated skin as determined by methods known in the art. In some embodiments, the level of TEWL of treated skin is statistically lower compared to baseline levels as determined by methods known in the art. In some embodiments, TEWL is measured by a Tewameter™ 300 meter (Courage-Khazaka Electronics). In some embodiments, TEWL is measured by a Tewameter™ Nano (Courage-Khazaka Electronics). In some embodiments, TEWL is measured by a Tewameter Triple™ 330T (Courage-Khazaka Electronics). In some embodiments, TEWL is measured by an Invitro Measured by Tewameter VT310 (Courage-Khazaka Electronics). In some embodiments, the TEWL of treated skin is >10% lower on day 7 after administration of the system.

[0156] In some embodiments, the skin is assessed for improved skin smoothness. In some embodiments, the skin after administration of the system is smoother than the skin before administration of the system. In some embodiments, the skin smoothness is assessed using Surface Evaluation of Living Skin (SELS). In some embodiments, In some cases, the skin is assessed using the phaseshift rapid in vivo measurement of skin (PRIMOS). In embodiments, the skin is assessed by 3D skin profilometry via Canfield Primos. In some embodiments, the skin is assessed using the Lemperle wrinkle scale. In some embodiments, the skin is assessed using the 7-point subject satisfaction scale. In some embodiments, the skin is assessed using the corners of the mouth. In some embodiments, the skin is assessed using the Allergan Skin Roughness scale.

[0157] In some embodiments, the skin is assessed to determine the presence and / or extent of crosslinked material in the skin. In some embodiments, the presence and / or extent of crosslinked material in the skin is measured using near infrared (NIR) spectroscopy, confocal microscopy, integrating sphere spectrophotometers, or determined using viscoelastic deformation (VED, mm), elastic deformation (ED, mm), limit deformation (UD, mm), and pressure-deformation ratio methods. In some embodiments, the presence and / or extent of crosslinked material in the skin is measured by applying ultraviolet light to a target site. In some embodiments, the presence and / or extent of crosslinked material in the skin is determined by observing fluorescence upon application of ultraviolet light to a target site.

[0158] In some embodiments, the presence of the crosslinked material at the target site becomes observable after a period of time (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 30 days or more). In some embodiments, the presence of the crosslinked material at the target site becomes observable after about 2 days. In some embodiments, the presence of the crosslinked material at the target site becomes observable after about 9 days. In some embodiments, the presence of the crosslinked material at the target site becomes observable after about 11 days. In some embodiments, the presence of the crosslinked material at the target site becomes observable after about 20 days. In some embodiments, the presence of the crosslinked material at the target site becomes observable after about 30 days. EXAMPLES

[0159] The present examples describe, inter alia, certain strategies that can be used to characterize and / or assess crosslinkable entities (and / or their constituent parts and / or combinations) as described herein, and / or crosslinked materials formed by their reactions. Such strategies (or their equivalents, as will be recognized by those of skill in the art upon reading this disclosure) can be used to assess potential crosslinkable entities, their constituent parts (e.g., moieties) or combinations, and / or the crosslinked materials (e.g., gels) they form, for suitability for use in accordance with the present disclosure. Thus, in some embodiments, the present disclosure provides techniques for characterizing and / or selecting useful moieties, crosslinkable entities, and / or combinations thereof. Example 1: Synthesis of crosslinkable entities Synthesis of the first crosslinkable entity: HA-gly-CBT Step 1 – CBT-Glycine [ka]

[0160] A mixture of Boc-glycine (1.4 eq.), DIPEA (2.1 eq.), and TBTU (1.4 eq.) in anhydrous DMF (approximately 40 mg Boc-glycine / mL) is stirred at room temperature for 30 min. A solution of CBT (1 eq.) in anhydrous DMF (approximately 100 mg CBT / mL) and DIPEA (1.4 eq.) is added to the mixture. The reaction is stirred at room temperature under nitrogen for 4 h. The solvent is removed by rotary evaporation, the crude product is dissolved in ethyl acetate, and the organic layer is washed successively with water, saturated aqueous sodium bicarbonate, and then brine. Boc-gly-CBT is then further isolated by flash chromatography using 3:1 to 1:1 hexanes:ethyl acetate. Boc deprotection is achieved by stirring Boc-gly-CBT in 1:1 TFA:DCM for 1 h. The solvent is then removed by rotary evaporation. Step 2 - HA-gly-CBT [ka]

[0161] After dissolving HA (1 eq.) in 1:1 DMSO:DI water (10 mg / mL), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS, 0.18 eq.), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 0.18 eq.), and gly-CBT (0.18 eq.) are added sequentially to the reaction mixture. After reacting for 24 h at room temperature, HA-gly-CBT is purified by repeated precipitation from ethanol, followed by dialysis for 24 h. HA-gly-CBT is then dried by lyophilization. The proton NMR spectrum of HA-gly-CBT is illustrated in FIG. 2.

[0162] Synthesis of HA-gly-CBT with different molecular weights HA-gly-CBT was synthesized with different degrees of functionality of CBT by varying the stoichiometry of the reactants. Additionally, HA-gly-CBT of different molecular weights was synthesized by starting with different molecular weights of HA (approximately 10 kDa, 50 kDa, and 250 kDa purchased from Creative PEG works) using the two-step synthesis described herein. To verify the synthesis of crosslinkable entities with different molecular weights, gel permeation chromatography (GPC) was used. GPC analysis was performed on an Agilent 1100 HPLC using a DAD and RI detector in series with an Agilent PL Aquagel-OH MIXED-M column (8 um 300 x 7.5 mm). A mobile phase of 0.2 M NaNO3 in water was used at a flow rate of 1 mL / min. The GPC analysis of the three crosslinkable entities prepared using 10 kDa, 50 kDa, and 250 kDa HA is illustrated in Figure 3.

[0163] Synthesis of different variants of HA-CBT In some embodiments, the first cross-linking entity may be an HA derivative functionalized with a CBT derivative. For example, in some embodiments, a CBT derivative (other than 6-amino-2-cyanobenzothiazole), such as 6-hydroxybenzothiazole-2-carbonitrile or another chemical derivative of the structure shown in Figure 4a, may be conjugated to HA. In some embodiments, the CBT derivative may be further derivatized with a chemical linker to facilitate conjugation to HA prior to conjugation with HA. In some embodiments, the chemical linker may be an amino acid, among other chemical groups. In some embodiments, the CBT derivative may be conjugated to a carboxylic acid (R1), an alcohol (R2), or another chemical group on HA, as shown in Figure 4b.

[0164] In some embodiments, the lipophilicity and / or skin penetration of the HA-CBT derivative can be increased by conjugating one or more other chemical moieties to the HA backbone, for example using one or more CBT derivatives that can be more hydrophobic than CBT itself (and / or than HA itself), among other chemical modifications. In some embodiments, the HA-CBT includes (e.g., is conjugated to) one or more chemical moieties (e.g., one or more hydrophobic moieties), such as, for example, a fatty acid.

[0165] In some embodiments, one or more moieties (e.g., hydrophobic moieties, e.g., CBT derivatives and / or fatty acid moieties) can be conjugated to alcohols (R2) on the backbone of HA. In some embodiments, before or after such conjugation, the HA can be oxidized to convert a fraction of the alcohols (R2) to aldehydes.

[0166] In some embodiments, conjugation of a moiety to HA (e.g., an alcohol on the HA backbone) can be via a linker, and in some such embodiments, the linker can be a hydrophobic amino acid or other hydrophobic chemical moiety.

[0167] In some embodiments, one or more moieties conjugated to HA (or other polymer moieties) as described herein may be modified (e.g., functionalized) before or after such conjugation. In some embodiments, although this is just one particular embodiment, some or all (e.g., a fraction) of the CBT or CBT derivative moieties conjugated to HA may be further functionalized, for example, by reacting the nitrile group with a hydrophobic cysteine ​​derivative or another aminothiol, such as cysteamine. Synthesis of the second crosslinkable entity: Cys-PEG-Cys [ka]

[0168] Cys-PEG-Cys is synthesized by conjugating the carboxylic acid on cysteine ​​(Cys) to the amine on the PEG diamine. A mixture of N-Boc-S-trityl-D-cysteine ​​(2.4 eq.) and 1,1'-carbonyldiimidazole (CDI, 3.0 eq.) in anhydrous DMF (approximately 55 mg N-Boc-S-trityl-D-cysteine / mL) is stirred at room temperature for 30 minutes. A solution of PEG diamine (approximately 2000 Da, 1 eq.) in anhydrous DMF (approximately 125 mg PEG diamine / mL) is then added to the reaction mixture. After 4 hours of reaction at room temperature, the DMF is removed by rotary evaporation and the polymer is precipitated twice into 1:1 diethyl ether:n-hexane. Boc and trityl deprotection is achieved by adding the polymer to a 0.85:0.10:0.05 solution of TFA:triisopropylsilane:DI water (approximately 50 mg / mL) at room temperature. Cys-PEG-Cys is then purified by precipitating twice into 1:1 ice-cold diethyl ether:n-hexane. TFA is further removed by dissolving the polymer in 0.1 M HCl and removing the solvent by rotary evaporation. The polymer is then lyophilized to obtain Cys-PEG-Cys.

[0169] Synthesis of the third crosslinkable entity: Cys-EDA-Cys Lower molecular weight Cys-based crosslinkable entities can be prepared using ethylenediamine as the linker (Cys-EDA-Cys). Such materials were prepared by a procedure similar to that described above for Cys-PEG-Cys. A mixture of N-Boc-S-trityl-D-cysteine ​​(2.8 equiv.) and 1,1'-carbonyldiimidazole (CDI, 2.8 equiv.) in anhydrous DMF (approximately 55 mg N-Boc-S-trityl-D-cysteine / mL) was stirred at room temperature for 30 min. EDA was then added directly to the reaction mixture. After 3.5 hours of reaction at room temperature, the DMF was removed by rotary evaporation and the polymer was extracted into DCM and washed first with water and then with brine. The protected product was then isolated by flash chromatography using 1.5:1 ethyl acetate:hexane containing 0.05% triethylamine. Boc and trityl deprotection was achieved by adding the polymer to a 0.85:0.10:0.05 solution of TFA:triisopropylsilane:DI water (approximately 50 mg / mL) at room temperature and stirred for 4 hours. Cys-EDA-Cys was then purified by removing the solvent by rotary evaporation and extracting the product from DCM with DI water (2 times). The TFA was further removed by dissolving the polymer in 0.1 M HCl and removing the solvent by rotary evaporation. The polymer was then lyophilized before obtaining Cys-EDA-Cys. The proton NMR spectrum of Cys-EDA-Cys is illustrated in FIG. 6.

[0170] Synthesis of variants with different Cys bridging moieties In some embodiments, the second cross-linkable entity contains two or more aminothiols (i.e., two or more cross-linkable aminothiol moieties), each of which, at least in some embodiments, can be conjugated via a linker moiety.

[0171] In some embodiments, such aminothiols are 1,2-, 1,3-, or 1,4-aminothiols, or combinations thereof. In some embodiments, the aminothiols are D-cysteine, L-cysteine, derivatives of cysteine, or combinations thereof. In some embodiments, the aminothiols are capped with another chemical moiety that can be released to expose the aminothiols, for example, under physiological conditions.

[0172] In some embodiments, the linker may be or include a polymer, such as PEG, a diamine, or a polypeptide, hi some embodiments, the linker may be or include a small molecule or an amino acid, such as ethylenediamine or lysine. Example 2: Assessment of the characteristics of crosslinkable entities, their components and / or combinations, and / or the crosslinked materials they produce ex vivo

[0173] The present examples describe certain ex vivo strategies that can be used to characterize or assess the crosslinkable entities (and / or their constituent components and / or combinations) as described herein, and / or the crosslinked materials formed by their reactions.

[0174] viscosity In some embodiments, it may be desirable to assess the viscosity of a preparation of one or both crosslinkable entities separately (e.g., before the crosslinkable entities are contacted with each other and / or allowed to crosslink).

[0175] Those skilled in the art will be aware of techniques for assessing viscosity. By way of example only, those skilled in the art will be aware of various procedures for assessing viscosity, for example, utilizing a rheometer (e.g., having concentric cylinder, cone and plate, or parallel plate geometries).

[0176] As described herein, in some embodiments, preparations of individual crosslinkable moieties for use as described herein can have a viscosity low enough to permit delivery by injection (e.g., via a microneedle).

[0177] Rheological properties Thus, in some embodiments, crosslinkable entities (and / or their components and / or combinations) can be assessed for their usefulness as described herein based, in whole or in part, on one or more rheological properties of the crosslinked material they produce. In some embodiments, one or more rheological properties of the crosslinked material can be tailored based on the molecular weight (e.g., number average molecular weight), concentration, degree of substitution of the polymeric and / or crosslinking moieties, and the density and / or length of crosslinks within the material, etc.

[0178] Those skilled in the art will be familiar with techniques for assessing the rheological properties of materials crosslinked as described herein. For example, in some embodiments, rheological assessment can be performed using a rheometer AR-G2. Rheological flow and / or vibration analysis can be performed using Couette geometry (cup diameter=30.42 mm, gap=0.5 mm, and bob diameter and length of 27.95 mm and 42.15 mm, respectively). In some embodiments, it may be desirable to minimize or prevent solvent evaporation, for example, by thermally isolating the material to be assessed (solvent trapping) and checking its weight before and after the experiment (weight difference should desirably be less than 1%). Typically, the analysis is performed under constant temperature (37° C.). In some embodiments, gelation kinetics can be monitored via time sweep tests, and storage (G') and loss (G'') moduli can be measured as a function of time, for example, at a frequency of 10 rad / sec and 1% stress strain.

[0179] In some embodiments, the first and second crosslinkable entities can be premixed (e.g., for a short time, e.g., about 5 minutes), loaded, and measured at the test temperature. Either or both of the frequency and flow sweep tests can be performed after crosslinking has occurred between the crosslinkable entities. In some embodiments, the frequency sweep test can be performed isothermally, e.g., in a frequency range of 0.01<ω<100 rad / sec, while maintaining a stress strain of, e.g., 1%. Desirably, the normal force can be maintained at a constant value as well. In some embodiments, care should be taken to ensure that residual stresses resulting from the load are relaxed. In some embodiments, the crosslinked material can be given a soak time to equilibrate at the test temperature. In some embodiments, the flow sweep experiment can be performed at a frequency range of 0.0005<γ<100 s. -1 to determine the zero shear viscosity and / or aggregate shear thinning regime. In some embodiments, data points can be collected once every 100 seconds.

[0180] In some embodiments, materials crosslinked as described herein desirably have a storage modulus in the range of 50 Pa to 10 kPa. In some embodiments, materials crosslinked as described herein desirably have a storage modulus in the range of 10 Pa to 100 Pa. In some embodiments, materials crosslinked as described herein desirably have a storage modulus in the range of 100 Pa to 300 Pa. In some embodiments, materials crosslinked as described herein desirably have a storage modulus in the range of 300 Pa to 1000 Pa. In some embodiments, materials crosslinked as described herein desirably have a storage modulus in the range of 1 kPa to 10 kPa. In some embodiments, materials crosslinked as described herein desirably have a storage modulus in the range of 5 kPa to 10 kPa. In some embodiments, materials crosslinked as described herein desirably have a storage modulus greater than 10 kPa.

[0181] Gelation kinetics In some embodiments, the desired crosslinkable entities interact with each other to produce a crosslinked material with appropriate kinetics as described herein under physiologically relevant conditions (e.g., at 37° C. and / or in the presence of physiologically relevant salts and / or other agents). In some embodiments, it may be useful to assess gelation kinetics in vitro, for example, by combining a first crosslinkable entity and a second crosslinkable entity together under such physiologically relevant conditions and monitoring their interaction, and assessing, for example, the dynamic storage modulus (G') and loss modulus (e.g., G''). As will be recognized by one of skill in the art, the point at which the dynamic storage modulus exceeds the loss modulus can be considered the gelation time.

[0182] In some embodiments, the first and second crosslinkable entities are mixed in a container (e.g., a test tube) and after a period of time the test tube is inverted but the resulting mixture does not flow out of the container. In some such embodiments, the period of time is 10 seconds, 30 seconds, 60 seconds, 90 seconds, or 360 seconds.

[0183] In some embodiments, desirable crosslinkable entities (or components / portions or combinations thereof) are such that when combined ex vivo, they result in a gelation time, determined as described herein, of less than about 30 minutes.

[0184] In some embodiments, the appropriate or desired gelation time will depend on the method or mode of administration. For example, if the first and second crosslinkable moieties are combined or otherwise contacted with one another at a site other than the target gelation site (e.g., in a syringe or at the site of administration), it is typically desirable for the gelation time to be long enough to prevent significant crosslinking away from the target gelation site.

[0185] Swelling In some embodiments, crosslinkable entities, components or combinations thereof can be assessed for usefulness in accordance with the present disclosure by determining one or more swelling characteristics of the crosslinked materials they produce. In some embodiments, the degree of swelling can be determined by comparing the degree of swelling of the crosslinked material in a dry form with the degree of swelling of the crosslinked material in a fully swollen form (e.g., in equilibrium with an external aqueous buffer: QM swollen / M dry ) based on the mass ratio between the crosslinked polymer moieties and the crosslinked polymer moieties. In some embodiments, one or more swelling properties of the crosslinked material can be tailored based on the molecular weight (e.g., number average molecular weight), concentration, degree of substitution of the polymer moieties and / or crosslinked moieties, and the density and / or length of crosslinks within the material, etc. In some embodiments, materials crosslinked as described herein can have a swelling ratio in the range of 50 to 1000.

[0186] Hyaluronidase resistance One of skill in the art will recognize that cross-linking can increase the resistance of biopolymers to degradation, increasing their durability in situ. To demonstrate this, HA gels were formed ex vivo and exposed to hyaluronidase, an enzyme naturally found in the body that degrades HA.

[0187] HA-gly-CBT (50 μL, 15 mg / mL, PBS) was mixed with either PBS (20 μL) or Cys-PEG-Cys (20 μL, 20 mg / mL) and incubated at 37° C. for 1.5 h. The mixture of HA-gly-CBT and PBS remained a viscous liquid, while the mixture of HA-gly-CBT and Cys-PEG-Cys formed a clear gel. To each formulation, bovine testicular hyaluronidase (5 μL) was added and the mixture was gently vortexed to obtain a final enzyme concentration of 100 units / mL. At the defined time points, samples were centrifuged to remove insoluble gel components, and aliquots of each of the supernatants were analyzed. Resistance to degradation was assessed by summing the fraction of remaining soluble hyaluronan with a high molecular weight (determined by GPC analysis of the supernatant) and the fraction of remaining insoluble hyaluronan (determined by GPC analysis of the supernatant using a plate reader).

[0188] The degradation of HA-gly-CBT mixed with PBS was significantly more rapid than that of cross-linked HA-gly-CBT (HA-gly-CBT mixed with Cys-PEG-Cys). After 24 hours, less than 15% of the HA-gly-CBT remained as high molecular weight in the uncross-linked sample, while >50% of the cross-linked HA-gly-CBT remained as a gel. Images of the gel after 24 hours of incubation are shown in FIG. 9 and confirm the demonstrated resistance to degradation by hyaluronidase.

[0189] In some embodiments, the degree of crosslinking in the crosslinked material is sufficient to impart durability in situ, and in some such embodiments, the crosslinked material exhibits increased durability relative to a suitable reference, such as a corresponding uncrosslinked material. Example 3: Formulation of cross-linkable entities

[0190] The following subsections (i.e., Sections 3.1-3.11) provide exemplary formulations, each of which includes at least one crosslinkable entity. In some embodiments, the component labeled "crosslinkable entity" can be substituted for a different crosslinkable entity as appropriate, as provided herein. In some embodiments, the system includes a first formulation and a second formulation of Sections 3.1-3.11, which are paired together so long as the crosslinkable entity of the first formulation can react with the crosslinkable entity of the second formulation to form a crosslinked material.

[0191] 3.1. Exemplary serum A serum (which may be utilized, for example, as an anti-aging serum) may be prepared by combining Parts A and B as outlined in Table 3.1, and then adjusting the pH of the serum to about 5.0 to about 6.0. Prepare by adding Part C up to volume of 5.5.

[0192] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in serum preparations as suggested below. [Table 1]

[0193] 3.2. Exemplary Lotions A lotion formulation (e.g., can be used as a soothing and / or moisturizing lotion) is prepared as outlined herein. Part A is prepared by combining the ingredients outlined in Table 3.2 and heating to 75°C with mixing. Separately, Part B is prepared by combining the ingredients outlined in Table 3.2 and heating to 75°C with mixing. Using high shear mixing, Part B is added to Part A and mixed at 75°C. The formulation is cooled with moderate mixing. Part C is mixed at 35°C until uniform. Part D is added to adjust the pH to 5.0±0.5.

[0194] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in lotion formulations as suggested below. [Table 2]

[0195] 3.3. Exemplary Lotions A lotion formulation (which can be used, for example, as a natural sunscreen lotion for the face) is prepared as outlined herein. Part A is prepared by combining the ingredients outlined in Table 3.3 and heating to 85°C. Part B is prepared by combining the ingredients outlined in Table 3.2 and heating to 85°C. Part C is added to Part A with stirring and maintain temperature. Part B is slowly added to Part A / C with high shear stirring. The mixture is homogenized. The formulation is stirred, cooled to room temperature and Part D is added below 40°C.

[0196] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in lotion formulations as suggested below. [Table 3]

[0197] 3.4. Exemplary W / O Emulsions An emulsion formulation (which can be used, for example, as a soothing w / o cream) is prepared as outlined herein. Part A is prepared by mixing the ingredients outlined in Table 3.4 until dissolved. The ingredients of Part B are then added to Part A using high shear to obtain the formulation.

[0198] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in emulsion preparation as suggested below. [Table 4]

[0199] 3.5. Exemplary Hydrogel Masks A hydrogel mask formulation (which can be used, for example, as a solid fiber mask with impregnated serum) is prepared as outlined herein. Part A is prepared by dissolving tetrasodium EDTA in water under propeller mixing. Gum is pre-dispersed in glycerin to create a slurry. The slurry is added to the tetrasodium EDTA solution under propeller mixing and mixed until uniform and free of lumps. Part A is heated to 85-90°C to hydrate the gum. While Part A is warm, Part B as outlined in Table 3.5 is added with mixing.

[0200] The ingredients of Part C as outlined in Table 3.5 are premixed and added to Parts A and B. The warm solution is immediately poured into a mold and allowed to sit undisturbed. Once the gel has cooled and settled, the formulation is appropriately packaged.

[0201] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in the hydrogel mask formulation as suggested below. [Table 5]

[0202] 3.6. Exemplary powders for creaming serums A serum formulation (which can be used as a BB (beauty balm) powder to cream a soothing serum, for example) is prepared as outlined herein. Procedure: Add all ingredients of Part A as outlined in Table 3.6 and mix at high shear until a homogenous mixture is obtained. Add Part B as outlined in Table 3.6 and powder the ingredients and mix until a free-flowing powder is obtained.

[0203] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in serum preparations as suggested below. [Table 6]

[0204] 3.7. Exemplary Solid Formulations A solid formulation (which can be used, for example, as a solid stick to target deep wrinkles) is prepared as outlined herein. Procedure: Heat Part A as described in Table 3.7 to 95°C, mix until dissolved, then add to Phase B as described in Table 3.7, heat to 95°C, cool to 75°C, add Phase C. Mix until uniform and pour into moulds or containers.

[0205] Freezing point: 70℃

[0206] Appearance: Light amber transparent to translucent gel

[0207] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in the solid preparation as suggested below. [Table 7-1] [Table 7-2]

[0208] 3.8. Example of Mist A mist formulation (which can be used, for example, as a revitalizing mist with antioxidants at pH 5) is prepared as outlined herein. Combine ingredients as outlined in Table 3.8 to obtain Part A. Combine ingredients as outlined in Table 3.8 to obtain Part B. Add Part A to Part B while mixing until uniform. Add Part C to the mixture of Parts A and B. Mix the resulting mixture until uniform. Adjust pH to a pH of 4.0-5.0 for stability. Package the formulation into a spray bottle. [Table 8]

[0209] 3.9. Exemplary Serum Combine the ingredients in Part A as outlined in Table 3.9 and mix until homogenous. Combine the ingredients in Part B as outlined in Table 3.9 and add to Part A and mix until homogenous. Add Part C. If necessary, add Part D until the pH is adjusted to 5.0-5.5 with 25% citric acid solution.

[0210] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in serum preparations as suggested below. [Table 9]

[0211] 3.10. Exemplary Powders A powder formulation (e.g., can be used as a powder to cream the formulation with an inhibitor to slow / prevent cross-linking in the product until it is in the skin) is prepared as outlined herein. Combine the ingredients in Part A as outlined in Table 3.10 and mix at high shear until a homogenous mixture is obtained. Add the powder ingredients in Part B and mix until a free-flowing powder is obtained. Combine Part C and mix until homogenous.

[0212] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in serum preparations as suggested below. [Table 10]

[0213] 3.11 Exemplary Lotion A lotion (which can be used, for example, as a liquid crystal solid nanoparticle dual action lotion) is prepared as outlined herein. The components of Part A as outlined in Table 3.11 are mixed and heated to 50°C. The components of Part B are added and the mixture is heated to 65°C-70°C. The components of Phase C as outlined in Table 3.11 are mixed at room temperature. The heated components of Parts A and B are then mixed together in a vessel under laminar flow conditions, thereby creating a lyotropic liquid crystalline mixture phase (AB) having an average particle size of 150 nm. The lyotropic liquid crystalline mixture Parts A and B are then mixed with Part C to form a dispersion. Once homogeneous, Phase D is added, the pH is adjusted by adding Phase E with gentle mixing, and then cooled to room temperature.

[0214] Without wishing to be bound by any particular theory, Applicants note that the exemplary components may, in some embodiments, be functionally involved in serum preparations as suggested below. [Table 11]

[0215] Example 4: Assessment of the characteristics of crosslinkable entities in porcine skin, their components or combinations, and / or the crosslinked materials they produce

[0216] The present examples describe the use of pigskin studies to characterize or assess the crosslinkable entities (and / or their components and / or combinations) and / or crosslinked materials formed by their reactions as described herein.

[0217] In some embodiments, a system comprising a first crosslinkable entity and a second crosslinkable entity is applied to the pigskin. In some such embodiments, the application of the first crosslinkable entity is separated in time (i.e., a period of time) from the application of the second crosslinkable entity. In some embodiments, a number of different periods of separation can be assessed. Those skilled in the art will recognize that the period of time can depend on the desired penetration depth, skin type, and target location. In some such embodiments, a number of different periods of separation can be assessed as a function of the desired penetration depth, skin type, and target location.

[0218] In some embodiments, the permeability of the first and / or second crosslinkable entities in the pig skin is assessed at one or more time points after administration of one or both of the crosslinkable entities. For example, in some embodiments, such assessment is performed at one or more time points selected from the group consisting of 1 hour, 4 hours, 12 hours, 24 hours, and 48 hours.

[0219] In some embodiments, one or more pig skin samples can be thawed, for example at room temperature (e.g., for 30 minutes or longer), prior to use. In some embodiments, hair can be clipped from such samples (e.g., using scissors) and the samples can be rinsed, for example, using 0.9% saline.

[0220] In some embodiments, subcutaneous in situ gelation can be assessed by administering a first crosslinkable entity and a second crosslinkable entity to one or more sites on or within the sample. For example, in some embodiments, a solution of the first crosslinkable entity and the second crosslinkable entity can be injected into the sample (e.g., separately or as a mixture, optionally after a premix period, and optionally in one or more different concentration ratios). Alternatively or additionally, in some embodiments, pig skin samples can be excised to reach their underlying dermis layer, and the first and second crosslinkable moieties can be administered to fill the gaps.

[0221] In some embodiments, the skin sample administered with the first and second crosslinkable moieties can be maintained under stable conditions (e.g., stored at room temperature in a Petri dish) for a period of time to allow crosslinking (e.g., gelation) to occur in situ.

[0222] In some embodiments, the degree of crosslinking can be assessed at one or more time points after administration (e.g., after injection of the mixture, or after injection of the first crosslinkable moiety, or more likely after injection of the second crosslinkable moiety), e.g., by sectioning the sample (e.g., using a scalpel). In some embodiments, such sectioning involves preparing small (e.g., about 0.4 cm x 0.4 cm) squares, which can be frozen (e.g., by immersion in an isopentane / dry ice bath) and stored, e.g., at -80°C.

[0223] Whether or not such storage has taken place, thin (e.g., about 20 μm) skin sections can be obtained (e.g., using a cryostat) and imaged (e.g., using a confocal microscope).

[0224] Penetration of HA-gly-CBT into pig skin Frozen porcine skin was equilibrated to room temperature and cut into approximately 1.5 × 1.5 cm squares. Prior to incubation, one skin sample was tape-stripped 20 times to remove the stratum corneum (SC). All skin pieces were washed with PBS and then placed in Franz diffusion cells (1 cm) at 37°C for 5 min. 2 ) PBS or HA-gly-CBT (approximately 250 kDa, 10 mg / mL, PBS) (150 μL) was applied into the donor compartment, the donor compartment was sealed with parafilm, and the cells were incubated at 37° C. for 18 hours in a humidified oven.

[0225] The skin surface was washed three times with PBS in a Franz cell, and then a small area in the center of the applied area was excised with a scalpel. The skin tissue was placed in OCT in a biopsy mold and flash frozen by placing the mold in a methylpentane / dry ice slurry. The frozen skin was sectioned into 20 μm sections on a cryostat microtome. A drop of ProLong Gold was placed on each section and covered with a cover slip before imaging. Fluorescent images were taken with a Zeiss AxioPlan2 using a 10x objective with a DAPI filter (Zeiss filter 49) to visualize the location of the HA-gly-CBT.

[0226] Compared to skin treated with PBS alone, skin treated with HA-gly-CBT showed significantly higher fluorescence in the epidermis, indicating dermal penetration of HA-gly-CBT through the SC (Figure 10). Stronger fluorescence was observed in skin that was tape-stripped prior to topical application of HA-gly-CBT, indicating increased penetration after the top skin barrier is disrupted.

[0227] The validation of the penetration of intact HA-gly-CBT was assessed using gel permeation chromatography (GPC). HA-gly-CBT (15 mg / mL, 300 μL, 0.5% Tween 20(DI water containing 0.01% HA) was applied topically to six separate pig skin sections in Franz diffusion cells. The samples were incubated in a sealed donor compartment at 37°C for 18 hours, and the skin surface was washed three times with PBS. The stratum corneum was isolated from the samples using tape strips, then the epidermis was scraped off from the dermis, and the dermis was cut into small pieces. HA-gly-CBT was extracted from each of the skin sections (top 8 layers of SC, bottom 8 layers of SC, epidermis, and dermis) by incubating overnight at 37°C in 1:1 PBS:methanol. The volume of the extract was reduced using rotary evaporation, and the concentrated solution was analyzed by GPC (using the same conditions as described in Example 1, a mobile phase of 9:1 DI:MeOH, 0.2M NaNO3).

[0228] The GPC signals from each layer of the samples (stratum corneum, epidermis, dermis) were averaged across all six samples (Figure 11). Signals corresponding to HA-gly-CBT were observed in both the stratum corneum and epidermal / dermal extracts, indicating that intact HA-gly-CBT was delivered to each layer of the skin. To demonstrate the reproducibility of the results, error bars for the SC and epidermal / dermal extracts are included in Figures 11b and 11c, respectively.

[0229] In situ crosslinking in porcine skin To assess the biocompatibility of the crosslinking reaction between the two crosslinkable entities, the two components were introduced directly into porcine skin. HA-gly-CBT (30 μL, 5 mg / mL, PBS) was injected into two porcine skin samples just below the skin surface using a 31-gauge insulin syringe. One of the skin samples was then injected with Cys-PEG-Cys (20 mg / mL, PBS) (using the same technique) one hour after the injection of HA-gly-CBT. Both skin samples were incubated overnight at 37° C. in a humidified oven. After incubation, the samples were frozen and sectioned as described above.

[0230] Half of the sections from each group were washed with DI water at 37° C. for 1.5 hours to remove free HA-gly-CBT. Slides were imaged using a fluorescent microscope as described above. Skin fluorescence intensity in washed sections of skin injected with HA-gly-CBT alone was significantly lower compared to unwashed sections ( FIG. 12A ). However, skin samples injected with both crosslinkable entities showed similar skin fluorescence intensity in both washed and unwashed sections ( FIG. 12B ). These results suggest that the two crosslinkable entities reacted in the skin to form an insoluble gel that was not removed during the slide washing step. Example 5: Administration to humans

[0231] Examples of the present invention describe the administration of a system as described herein to a human, and the assessment of one or more skin characteristics in light of such administration.

[0232] As described herein, a first crosslinkable entity and a second crosslinkable entity are administered to a skin site. In some embodiments, both crosslinkable entities are administered to the same site, and in some embodiments, each can be administered to a different site, so long as both ultimately reach the intended target site, and thus the crosslinked material formed by the reaction of the first crosslinkable entity and the second crosslinkable entity is formed or otherwise provided in situ at the target site.

[0233] In some embodiments, one or both of the first crosslinkable entity and the second crosslinkable entity are administered to the target site (e.g., by injection). In some embodiments, one or both of the first crosslinkable entity and the second crosslinkable entity are administered to a site above (i.e., external to) the target site such that the crosslinkable entity must penetrate the skin tissue to reach the target site. In particular, the present disclosure provides crosslinkable entities that have certain characteristics that facilitate such penetration, such as, for example, lipophilicity and / or molecular weight (e.g., number average molecular weight).

[0234] In some embodiments, administration of a first crosslinkable entity is separated in time (i.e., a period of time) from administration of a second crosslinkable entity. In some such embodiments, the period of time is a period of time sufficient to allow substantial penetration of the first-administered crosslinkable entity to and / or into the target site before the second-administered crosslinkable entity is administered.

[0235] In some embodiments, multiple administrations are performed for at least one of the crosslinkable entities, and in some embodiments, multiple administrations are performed for each of the crosslinkable entities. In some embodiments, administration of both crosslinkable moieties is considered a "dose" of the provided system. In some embodiments, administration of one crosslinkable entity to a subject who has already received administration of the other crosslinkable entity is considered a "dose" of the provided system. In some embodiments, multiple doses are administered.

[0236] In some embodiments, the crosslinkable entity is the formulation of Example 3. In some embodiments, the formulation of the first crosslinkable entity and the formulation of the second crosslinkable entity are the same. In some embodiments, the formulation of the first crosslinkable entity and the formulation of the second crosslinkable entity are different.

[0237] Assessment of skin improvement In some embodiments, one or more characteristics of the skin (e.g., skin at and / or on a target site) are assessed after administration of the system (e.g., at one or more time points selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, and 31 days or longer after administration, e.g., after administration of any particular dose or complete dosing regimen). In some embodiments, one or more characteristics of the skin (e.g., skin at and / or on the target site) are assessed after administration of the system (e.g., at one or more time points selected from the group consisting of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months or longer after administration, e.g., after administration of any particular dose or complete dosing regimen). In some embodiments, one or more characteristics of the skin (e.g., skin at and / or on the target site) are assessed about 3.5 days after administration of the system. In some embodiments, one or more characteristics of the skin (e.g., skin at and / or on the target site) are assessed about 9 days after administration of the system.

[0238] For example, in some embodiments, one or more visual features of the skin are assessed following administration of the system (e.g., compared to prior to such administration). In some embodiments, the visual features are assessed by one or more expert graders with reference to the Global Aesthetic Improvement Scale (GAIS), which, as known in the art, is a standardized, validated 5-point scale for measuring response to cosmetic procedures that has been shown to have good intra- and inter-rater reliability, as well as intraclass correlation coefficients generally in the range of 0.85-0.95 (e.g., Carruthers & Carruthers, J. Cosmet Laser, 2003). (See, Therp. 12:235, 2010.) In some embodiments, administration of a system as described herein achieves an improvement of at least one grade on the GAIS scale.

[0239] Alternatively or additionally, in some embodiments, one or more skin features can be assessed using established scales, such as the Lemperle Wrinkle Scale, the Subject Satisfaction Scale (typically 7 points), the Allergan Skin Roughness Scale, or any other suitable scale. In some embodiments, any validated scale can be utilized that includes subjective and / or objective assessment of one or more physical, functional, or visual attributes of the skin (e.g., smoothness, radiance, firmness, elasticity, volume, etc.).

[0240] In some embodiments, the one or more skin features may be or relate to enhanced mechanical support of the dermal extracellular matrix, elongation and / or spreading of fibroblasts, upregulation of type II TGF-beta receptor or its downstream targets (e.g., type I collagen synthesis), upregulation of connective tissue growth factor, and / or combinations thereof (see, e.g., Quan et al., J Invest Dermatol. 133:658, 2013). Those skilled in the art are aware of strategies for assessing such features, including after injection into human skin (including damaged, e.g., photodamaged, human skin) (e.g., Wang et al Arch Dermotal. 143:155. 2-7).

[0241] In some embodiments, skin hydration can be assessed after administration of the provided system. In some such embodiments, skin hydration is measured, for example, using a corneometer (e.g., a corneometer CM825, which provides a conductance measure of relative water content in the skin). In some embodiments, administration of the system as described herein achieves a statistically significant improvement in skin hydration, and in some such embodiments, such improvement is at least 10%, 15%, 20%, 25% or higher, for example, when measured at 12 hours, 24 hours, 36 hours or 48 hours after administration, as described herein. For example, when crosslinked material (HA-gly-CBT+Cys-PEG-Cys) is administered to the sample, a 26% improvement in skin hydration is observed immediately after application of the material. Furthermore, in clean, undamaged skin, long-lasting skin hydration is observed for more than 72 hours (as assessed using a Courage+Khazaka Corneometer® CM825).

[0242] A 20 mg / ml HA-gly-CBT solution was prepared in deionized water, polysorbate 20, and glycerin. A second Cys-PEG-Cys solution (20 mg / mL) in PBS was prepared and the pH was adjusted to 5.5-6.5 with NaOH. The HA-gly-CBT solution (20 μl) and the benchmark commercial HA serum solution (20 μl) were applied to a 3 cm x 3 cm area on the inner forearm immediately after the baseline reading. Further Corneometer readings were taken at 0.5 and 6 hours. After 6 hours, Cys-PEG-Cys solution (20 μl) was applied to the HA-gly-CBT test site and phosphate buffer (20 μl) was applied to the benchmark commercial HA serum site. Further Corneometer readings were then taken at 12, 24, 48, 72, and 96 hours. The results are tabulated in Table 3.12.

[0243] The benchmark commercial HA (hyaluronic acid) serum was a blend of high and low molecular weight hyaluronic acids containing 1.5% pure hyaluronic acid, described by the manufacturer as capable of rehydrating and plumping the skin to produce a younger, suppler appearance. The ingredients list for the benchmark commercial HA serum included water, glycerin, hydroxyethylpiperazine ethanesulfonic acid, sodium hyaluronate, PEG-60 hydrogenated castor oil, cereale seed extract / rye seed extract, calcium pantothenate, dipeptide diaminobutyroyl benzylamide diacetate, ascorbyl glucoside, disodium EDTA, pentylene glycol, phenoxyethanol, and chlorphenesin. [Table 12]

[0244] In some embodiments, the smoothness of skin can be assessed after administration of the provided system.In some such embodiments, the smoothness of skin is measured, for example, by 3D skin profilometry, for example, by using Canfield Promos device, and / or by silicon / epoxy cast scanning.In some embodiments, administration of the system as described herein achieves a statistically significant increase in smoothness of skin, and / or an increase in smoothness over time (with or without further administration).For example, the visual improvement of smoothness of skin after administration of the provided system is demonstrated using a 7-point skin smoothness improvement scale.As shown in FIG. 13, 2 days after application of the system, a 2-grade improvement is observed.

[0245] In some embodiments, the thickness of the skin can be assessed after administration of the provided systems. In some such embodiments, the thickness of the skin can be assessed, for example, by ultrasound profiling (see, e.g., Van Mulder et al. Vaccine 35:1810, 2017). In some embodiments, the thickness of the skin can be assessed by administration of the systems as described herein. Administration of Stem achieves a statistically significant increase in skin thickness.

[0246] In some embodiments, the relevant skin may be damaged (e.g., due to a disease, disorder or condition, or surgical, cosmetic or dermatological treatment), and improvements to such damaged skin are assessed. In some embodiments, damaged skin may be or include skin that has been treated with a laser (e.g., fractional laser), skin that has been treated with intense-pulsed-light, skin that has been treated with microneedles, skin that has been treated with a chemical peel, skin that has been exposed to skin abrasion, skin that has been damaged by trauma (including by surgery), skin that is affected by a medical condition, such as skin atrophy, extreme dryness, etc.

[0247] Alternatively or additionally, in some embodiments the skin of interest may be skin that is wrinkled or otherwise shows signs of aging, or may be skin where additional plumping and / or resilience is desired.

[0248] In some particular embodiments, the skin of interest may be or include skin on the subject's face in an area selected from the group consisting of the entire face and / or specific targets, such as both lips, the lower lip, the upper lip, dark circles under the eyes or other areas around the eyes (e.g., crow's feet), nasolabial folds, the forehead, the cheeks, the backs of the hands, the earlobes, the knees, the neck, the décolleté, the arms, the legs, the torso, the buttocks, the feet, and combinations thereof, including, for example, the entire face.

[0249] Infiltration assessment In some embodiments, it may be desirable to assess the penetration of one or both crosslinkable entities administered to a human as described herein.

[0250] In some embodiments, a biopsy is obtained of the site where the provided system has been administered or delivered, and histological examination of the biopsy is performed to assess the degree (e.g., amount) and / or depth of penetration. In some embodiments, the crosslinkable entity and / or crosslinked material is detected in or under the epidermis, dermis, or underlying subcutaneous tissue. In some embodiments, a significant percentage (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) of the amount of crosslinkable entity administered (e.g., to the skin surface and / or to a site external to the target site) is detected within the skin of the biopsy (e.g., on, in, in or under the epidermis, dermis, or underlying subcutaneous tissue, and / or otherwise at the target site).

[0251] Penetration of HA-gly-CBT into damaged human skin In some embodiments, the penetration of crosslinkable entities into human skin can be increased by skin barrier disruption techniques. For example, the ex vivo penetration of HA-gly-CBT (tagged with AlexaFluor 647 hydrazide) into human skin pretreated with various techniques was assessed by microscopy. Prior to application of the material, one skin sample was tape stripped 25 times to remove most of the stratum corneum. In the second sample, HA-gly-CBT was applied to the skin surface, and then the skin was treated with 40 rolls of a derma roller (500 μm needle) on the product. Both samples were incubated in a Franz cell in a humidified oven at 37° C. for 20 hours after topical application. Samples were sectioned at 20 μm as described above and imaged with a Cy5 filter under a fluorescent microscope (FIG. 14).

[0252] Significant fluorescent signals were observed in the epidermis and dermis of human skin that was tape-stripped prior to topical application of HA-gly-CBT. In samples treated with HA-gly-CBT and a dermaroller, significant fluorescent signals were observed in the epidermis, but only faint signals were observed in the dermis. Together, these examples illustrate the extent to which disruption of the human skin barrier can facilitate deeper penetration of HA-gly-CBT into human skin.

[0253] The ability of dermarolling to promote topical penetration of HA-gly-CBT was further investigated by applying a 50 kDa variant of HA-gly-CBT. HA-gly-CBT (50 kDa, tagged with AlexaFluor 647 hydrazide) was applied topically to human skin and rolled with a dermaroller (500 μm needle) for 20 times. The skin was transferred to a Franz cell and incubated at 37° C. for 20 hours. The skin was frozen, sectioned, and imaged with a fluorescent microscope using a Cy5 filter to observe the penetration depth (FIG. 18A). Two additional human skin samples were treated similarly. The stratum corneum was removed from the samples by tape stripping (20 times), and the epidermis and dermis were isolated with a scalpel. After extraction of HA-gly-CBT from each skin layer into 1:1 PBS:methanol (overnight at 37°C), the concentration of HA-gly-CBT was quantified by measuring the fluorescence of the AlexaFluor dye in each extract using a plate reader (Figure 18B).

[0254] Significant amounts of HA-gly-CBT (50 kDa) were detected in both the epidermis and dermis after treatment with the dermaroller. The observed penetration of HA-gly-CBT suggests that local delivery of stable HA-based crosslinked materials to the deep dermis may be feasible when combined with subsequent, simultaneous or prior topical delivery of cysteine-based crosslinkable entities. Example 6: Exemplary System

[0255] The administration sites on the skin of the subject's left inner forearm (see FIG. 15) were prepared for administration of the crosslinkable entity. The sites were prepared by cleaning with mild soap and warm water for 30 seconds, rinsing the sites with warm water for an additional 30 seconds, and gently patting the sites dry. Translucent Scotch tape was repeatedly applied and removed to each site until each was slightly glossy (approximately 40 cycles).

[0256] The first formulation was administered to each of the sites. [Table 13]

[0257] Upon administration of the first composition, each site is covered with a Tegaderm™ type film approximately 3 cm thick. 3 The area was covered with 10 ml of 10 ...

[0258] A second crosslinkable formulation was administered to each of the sites. [Table 14]

[0259] For administration of the second composition, each site is covered with a Tegaderm™ type film approximately 3 cm thick. 3 The site was left covered overnight. The Tegaderm™ paper was removed. The site was allowed to equilibrate for 10 minutes. At that time, the administration site was examined using visible and ultraviolet (approximately 365 nm) light. No recordable fluorescent signal was observed at sites A and B. A fluorescent signal was clearly visible at site C (see FIG. 15), indicating that the crosslinked material had penetrated the skin.

[0260] The administration sites were washed at least once daily with mild soap and water and gently wiped dry. The administration sites were left dry at all other times.

[0261] The visual appearance of the sites was photographed daily under both visible and UV light (approximately 365 nm). No recordable fluorescent signal was observed in sites A and B. A fluorescent signal was clearly visible in site C, indicating that the crosslinked material had penetrated the skin and was still present (FIG. 16).

[0262] Eleven days after administration of the system, the sites were inspected again using ultraviolet light. A fluorescent signal was clearly visible at site C, indicating that the cross-linked material had penetrated the skin and was still present after 11 days. Furthermore, the subject did not show any evidence of tolerability issues. The administration sites showed improved skin texture 11 days after administration compared to the sites before administration of the system. Example 7: Histological Measurement

[0263] The following crosslinkable entities were administered in a 3×3 cm square on the left inner forearm: 1.75 μL of 2% HA-gly-CBT 2.2 hours have passed 3.75 μL of 2% Cys-PEG-Cys 4.3 hours have passed 5.75 μL of 2% HA-gly-CBT 6.2 hours have passed 7.75 μL of 2% Cys-PEG-Cys

[0264] Tegaderm™ was applied on top of the application site between and after each application of the crosslinkable entity. The application process was repeated daily for two days, for a total of four applications of both HA-gly-CBT and Cys-PEG-Cys. On the morning of the third day, the site was cleaned and sterilized, after which a 4 mm biopsy was taken in the center of the application square. The biopsy was immediately thawed in OCT.

[0265] Biopsies were sectioned on a cryostat and sections were fixed on slides in neutral buffered formalin. Slides were then stained with Alcian Blue to detect hyaluronic acid and Nuclear Fast Red as a counterstain (Figure 17). Some slides were also pre-treated with hyaluronidase to remove hyaluronic acid before staining. As can be seen in Figure 17, there is a strong signal in the upper epidermis and dermis for slides that were not pre-treated with hyaluronidase. The present invention provides, for example, the following items. (Item 1) A system comprising a first crosslinkable entity and a second crosslinkable entity, (i) the first cross-linkable entity comprises a polymer conjugated to a first cross-linking moiety, the polymer being optionally conjugated to the first cross-linking moiety via a first linker; and (ii) the second cross-linkable entity comprises one or more second cross-linking moieties; The system is characterized by: (Item 2) 2. The system of claim 1, wherein the polymer is a hyaluronic acid (HA) polymer. (Item 3) 2. The system of claim 1, wherein the first bridging moiety is selected from the group consisting of cyanobenzothiazole (CBT) and analogs and derivatives thereof. (Item 4) 2. The system of claim 1, wherein the second cross-linking moiety is a divalent or polyvalent aminothiol, or a derivative thereof. (Item 5) 5. The system of claim 4, wherein the aminothiol is selected from the group consisting of D-cysteine, L-cysteine, and analogs and derivatives thereof. (Item 6) 2. The system of claim 1, wherein the first linker is an amino acid. (Item 7) 7. The system according to item 6, wherein the amino acid is glycine. (Item 8) 2. The system of claim 1, wherein the second crosslinkable entity comprises the second crosslinking moiety, a hydrophobic moiety, or a combination thereof, conjugated to one or more polymer moieties. (Item 9) 9. The system of claim 8, wherein the second cross-linkable entity comprises the second cross-linking moiety conjugated to a polymer. (Item 10) 10. The system of claim 9, wherein the polymer is selected from the group consisting of PEG, PEG-diamine, polyacrylic acid, N-(2-hydroxypropyl) methacrylamide (HPMA), polycaprolactone (PCL), and poly(lactic-co-glycolic acid) (PLGA). (Item 11) 11. The system of claim 10, wherein the polymer is PEG. (Item 12) 2. The system of claim 1, wherein the second cross-linkable entity comprises the second cross-linking moiety conjugated to a polypeptide. (Item 13) 2. The system of claim 1, wherein the second cross-linkable entity comprises the second cross-linking moiety conjugated to a small molecule or an amino acid. (Item 14) 14. The system of claim 13, wherein the second cross-linkable entity comprises the second cross-linking moiety conjugated to ethylenediamine, or a derivative or analog thereof. (Item 15) 14. The system of claim 13, wherein the second cross-linkable entity comprises the second cross-linking moiety conjugated to lysine, or a derivative or analog thereof. (Item 16) (i) a polymer conjugated to one or more cross-linking moieties selected from the group consisting of CBT, and derivatives and analogs thereof; and (ii) Di- or poly-aminothiols Including, the system. (Item 17) Item 17. The system of item 16, wherein the polymer is HA. (Item 18) 17. The system of claim 16, wherein the aminothiol is selected from the group consisting of D-cysteine, L-cysteine, and derivatives and analogs thereof. (Item 19) 17. The system of claim 16, wherein the polymer is conjugated to the one or more cross-linking moieties via a linker. (Item 20) 20. The system of claim 19, wherein the linker is an amino acid. (Item 21) 21. The system according to item 20, wherein the amino acid is glycine. (Item 22) 17. The system of claim 16, wherein the aminothiol is conjugated to a polymer. (Item 23) 23. The system of claim 22, wherein the polymer is PEG. (Item 24) 17. The system of claim 16, wherein the aminothiol is conjugated to a polypeptide. (Item 25) 17. The system of claim 16, wherein the aminothiol is conjugated to a small molecule or an amino acid. (Item 26) 26. The system of claim 25, wherein the aminothiol is conjugated to ethylenediamine, or a derivative or analog thereof. (Item 27) 26. The system of claim 25, wherein the aminothiol is conjugated to lysine, or a derivative or analog thereof. (Item 28) A system comprising a first crosslinkable entity and a second crosslinkable entity, (i) each entity: (a) containing a cross-linking moiety having a Log(P) in the range of 0 to 6; or (b) characterized by sufficient lipophilicity to achieve penetration of at least 50 microns into the skin; (ii) the first crosslinkable entity and the second crosslinkable entity, when combined in vitro under physiological conditions, react with each other in a period of less than about 60 minutes at 37° C., (a) Storage modulus in the range of 50 Pa to 10 kPa forming a crosslinked material characterized by (iii) each of said crosslinkable entities, when administered to a site on the surface of the skin, improves thickness, aesthetics, moisture content, smoothness, elasticity, radiance, fine lines, skin color, skin clarity, and combinations thereof; A system characterized in that a statistically significant improvement in a skin trait selected from the group consisting of: a combination of: is observed within 24 hours, 3 days, 7 days, 30 days, 60 days, or 90 days. (Item 29) A system comprising a first crosslinkable entity and a second crosslinkable entity, (i) each entity: (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to intact skin; or (b) characterized by sufficient lipophilicity to achieve penetration of at least 50 microns into the skin; (ii) the first crosslinkable entity and the second crosslinkable entity, when combined in vitro under physiological conditions, react with each other in a period of less than about 60 minutes at 37° C., (a) a storage modulus in the range of 50 Pa to 10 kPa, or (b) a molecular weight greater than the sum of the molecular weights of each individual crosslinkable entity forming a crosslinked material characterized by (iii) when said crosslinkable entity is administered to a site on the skin surface, a statistically significant improvement in a skin attribute selected from the group consisting of thickness, aesthetics, moisture content, smoothness, elasticity, radiance, fine lines, skin color, skin clarity, and combinations thereof is observed within 24 hours, 3 days, 7 days, 30 days, 60 days, or 90 days. (Item 30) A system comprising a first crosslinkable entity and a second crosslinkable entity maintained separate from each other, At least the first crosslinkable entity is comprising a polymer moiety conjugated to a cross-linking moiety; (a) contains a cross-linking moiety having a Log(P) in the range of 0 to 6; or (b) A system characterized by sufficient lipophilicity to achieve penetration of at least 50 microns into the skin. (Item 31) A system comprising a first crosslinkable entity and a second crosslinkable entity maintained separate from each other, At least the first crosslinkable entity is comprising a polymer moiety conjugated to a cross-linking moiety; (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to intact skin; or (b) A system characterized by sufficient lipophilicity to achieve penetration of at least 50 microns into the skin. (Item 32) The first crosslinkable entity and the second crosslinkable entity are (i) when combined in vitro under physiological conditions, react with each other in a period of less than about 60 minutes at 37°C; (a) Storage modulus in the range of 50 Pa to 10 kPa forming a crosslinked material characterized by (ii) The system of item 30, wherein each of the compositions, when administered to a site on the skin surface, is characterized in that a statistically significant improvement in a skin attribute selected from the group consisting of thickness, visible aesthetics (i.e., brightness, wrinkles, skin color, etc.), moisture content, tactile aesthetics (i.e., smoothness, plumpness, etc.), elasticity, and combinations thereof, is observed within 24 hours, 3 days, 7 days, 30 days, 60 days, or 90 days. (Item 33) The first crosslinkable entity and the second crosslinkable entity are (i) When combined in vitro under physiological conditions, the mixture is incubated at 37°C for less than about 60 minutes. They react with each other within a period of time, (a) a storage modulus in the range of 50 Pa to 10 kPa, or (b) a molecular weight greater than the sum of the molecular weights of each individual crosslinkable entity forming a crosslinked material characterized by (ii) The system of item 30, wherein each of the compositions, when administered to a site on the skin surface, is characterized in that a statistically significant improvement in a skin attribute selected from the group consisting of thickness, visible aesthetics (i.e., brightness, wrinkles, skin color, etc.), moisture content, tactile aesthetics (i.e., smoothness, plumpness, etc.), elasticity, and combinations thereof, is observed within 24 hours, 3 days, 7 days, 30 days, 60 days, or 90 days. (Item 34) 33. The system of claim 30 or 32, comprising a container having separate containers or compartments, each of the first crosslinkable entity and the second crosslinkable entity being disposed within one of the separate containers or compartments. (Item 35) Item 35. The system of item 34, wherein the container comprises a box having at least two separate containers disposed therein. (Item 36) Item 35. The system of item 34, wherein the container comprises a receptacle having at least two separate compartments therein. (Item 37) Item 37. The system of item 36, wherein the container is arranged and constructed such that the first crosslinkable entity and the second crosslinkable entity are combined during delivery. (Item 38) Item 37. The system of item 36, wherein the syringe is arranged and constructed such that the first crosslinkable entity and the second crosslinkable entity are maintained separate during delivery. (Item 39) 33. The system of claim 29 or 32, wherein the crosslinked material is further characterized by less than 20% mass loss upon degradation over 3 days in physiological buffer. (Item 40) 33. The system of claim 29 or 32, wherein the crosslinked material is further characterized by slower degradation of the crosslinked material when exposed to in vitro conditions simulating conditions for physiological degradation, as compared to degradation of one or both crosslinkable entities in the absence of crosslinking. (Item 41) 30. The system of claim 29, wherein at least one of the crosslinkable entities comprises a polymer moiety conjugated to a crosslinking moiety. (Item 42) Item 43. The system of item 41, wherein only one of the crosslinkable entities comprises a polymeric portion. The system of claim 30 or 41, wherein the polymer portion comprises a hyaluronic acid portion. (Item 44) 44. The system of claim 43, wherein the polymer moiety comprises a hyaluronic acid moiety and further comprises an additional moiety that increases lipophilicity. (Item 45) 30. The system of claim 29, wherein each of the crosslinkable entities comprises a polymer moiety conjugated to a crosslinking moiety. (Item 46) Item 46. The system of item 45, wherein each of the crosslinkable entities comprises the same polymer moiety. (Item 47) Item 46. The system of item 45, wherein each of the crosslinkable entities comprises a different polymer moiety. (Item 48) 30. The system of claim 29, wherein the bridging moiety is or comprises a moiety selected from the group consisting of cyanobenzothiazole (CBT), D-cysteine, L-cysteine, and analogs, derivatives, and combinations thereof. (Item 49) 30. The system of claim 29, wherein the cross-linking moiety is or comprises a moiety selected from the group consisting of diamines, peptides, dithiols, dihydrazides, and combinations thereof. (Item 50) Item 33. The system of item 29 or item 32, wherein the skin characteristic is thickness. (Item 51) Item 33. The system of item 29 or item 32, wherein the skin characteristic is moisture content. (Item 52) Item 52. The system of item 51, wherein the moisture content of the skin after administration of the first and second crosslinkable entities and water is higher compared to untreated skin. (Item 53) 53. The system according to claim 52, wherein the moisture content is higher than >20%. (Item 54) Item 54. The system according to item 53, wherein the moisture content is higher than >10%. (Item 55) Item 33. The system of item 29 or item 32, wherein the skin trait is improved as determined by a global aesthetic improvement measure. (Item 56) 56. The system of claim 55, wherein the skin trait is improved by >1 on the GAIS. (Item 57) Item 57. The system according to item 56, wherein the skin characteristic is improved by about 1 to about 3 with the GAIS. (Item 58) Item 33. The system of item 29 or item 32, wherein the skin characteristic is moisture content. (Item 59) Item 59. The system of item 58, wherein the moisture content of the skin is higher compared to untreated skin. (Item 60) 30. The system of claim 29, wherein each entity is characterized by a lipophilicity sufficient to achieve skin penetration that is greater than a crosslinkable entity lacking said crosslinking moieties. (Item 61) 30. The system according to item 29, wherein each entity is characterized by penetrating in vitro into and / or beneath the epidermis when applied ex vivo to intact skin. (Item 62) 30. The system according to item 29, wherein each entity is characterized by penetrating in vitro into and / or beneath the dermis when applied ex vivo to intact skin. (Item 63) 30. The system according to item 29, wherein each entity is characterized by penetrating in vitro into and / or beneath the epidermis when applied ex vivo to damaged skin. (Item 64) 30. The system according to item 29, each of which is characterized by penetrating into and / or beneath the dermis in vitro when applied ex vivo to damaged skin. (Item 65) 30. The system of item 29, wherein each entity is characterized by an ability to penetrate the skin to a depth of at least 50 microns. (Item 66) Item 66. The system of item 65, wherein each entity is characterized by an ability to penetrate the skin to a depth of at least one cell layer. (Item 67) 1. A method for improving skin characteristics comprising: administering separately to the skin a first crosslinkable entity and a second crosslinkable entity such that a crosslinked material is produced within the skin; (i) at least the first crosslinkable entity comprises a polymer moiety conjugated to a crosslinking moiety; (a) contains a cross-linking moiety having a Log(P) in the range of 0 to 6; or (b) is characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns; or (ii) each of the first crosslinkable entity and the second crosslinkable entity is (a) containing a cross-linking moiety having a Log(P) in the range of 0 to 6; or (b) characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns. (Item 68) 1. A method for improving skin characteristics comprising: administering separately to the skin a first crosslinkable entity and a second crosslinkable entity such that a crosslinked material is produced within the skin; (i) at least the first crosslinkable entity comprises a polymer moiety conjugated to a crosslinking moiety; (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to intact skin; or (b) is characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns; or (ii) each of the first crosslinkable entity and the second crosslinkable entity is (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to intact skin; or (b) characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns. (Item 69) 1. A method for improving skin characteristics comprising: administering separately to the skin a first crosslinkable entity and a second crosslinkable entity such that a crosslinked material is produced within the skin; (i) at least the first crosslinkable entity comprises a polymer moiety conjugated to a crosslinking moiety; (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to damaged skin; or (b) is characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns; or (ii) each of the first crosslinkable entity and the second crosslinkable entity is (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to damaged skin; or (b) characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns. (Item 70) Pre-treating or co-treating the skin with an agent or device that increases skin permeability. 70. The method of any one of items 67 to 69, further comprising the step of placing (Item 71) 71. The method of claim 70, wherein the medicament or device is or comprises a microneedle. (Item 72) Item 72. The method of item 71, wherein the microneedle contains the first or second crosslinkable entity. (Item 73) Item 73. The method of item 72, wherein the crosslinkable entity is disposed within the lumen of the microneedle. (Item 74) 74. The method of claim 72 or 73, wherein the crosslinkable entity is administered by injection through the microneedle. (Item 75) Item 75. The method of item 74, wherein the microneedles are formed from a material that dissolves or decomposes after application. (Item 76) Item 76. The method of item 75, wherein the crosslinkable entity is released upon dissolution or degradation of the microneedles. (Item 77) 1. A method for improving skin characteristics comprising: administering a first crosslinkable entity to a skin site such that a crosslinked material is produced within the skin; (i) the first entity, (a) contains a cross-linking moiety having a Log(P) in the range of 0 to 6; or (b) being characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns; waiting for a certain period of time; administering to the site a second crosslinkable entity reactive with the first crosslinkable entity; A method comprising: (Item 78) 1. A method for improving skin characteristics comprising: administering a first crosslinkable entity to a skin site such that a crosslinked material is produced within the skin; (i) the first entity, (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to intact skin; or (b) being characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns; waiting for a certain period of time; administering to the site a second crosslinkable entity reactive with the first crosslinkable entity; A method comprising: (Item 79) 70. The method of any one of items 67 to 69, wherein the separately administering steps comprise administering at different times such that a period of time separates administration of the first crosslinkable entity and administration of the second crosslinkable entity. (Item 80) The skin attribute is selected from the group consisting of thickness, aesthetics, moisture content, and smoothness. 80. The method according to item 67 or 77. (Item 81) 80. The method of claim 77 or 79, wherein the period of time is less than 20 minutes. (Item 82) 82. The method of claim 81, wherein the period of time is less than 10 minutes. (Item 83) Item 83. The method of item 82, wherein the period of time is less than 1 minute. (Item 84) Item 78. The method of item 77, wherein the first crosslinkable entity comprises a polymer portion and a crosslinking portion. (Item 85) The method of claim 67 or 84, wherein the polymer moiety is or comprises a glycosaminoglycan or polysaccharide. (Item 86) The method of claim 67 or claim 84, wherein the polymer portion comprises a synthetic polymer. (Item 87) 68. The method of claim 67, wherein the polymer moiety is or comprises a polypeptide. (Item 88) 85. The method of claim 67 or 84, wherein the polymer moiety has a size of about 10 to 250 kDa. (Item 89) 78. The method of any one of items 67 to 69 or item 77, further comprising administering water to the skin after administering the first crosslinkable entity and the second crosslinkable entity. (Item 90) 80. The method of any one of items 77, 78 or 79, further comprising the step of assessing the skin characteristics after administering the first crosslinkable entity and the second crosslinkable entity. (Item 91) 91. The method of any one of items 66 to 90, wherein the recited steps are repeated at least one time. (Item 92) Item 92. The method of item 91, wherein the steps are repeated once a day for several days. (Item 93) Item 92. The method of item 91, wherein the steps are repeated every 3 days. (Item 94) Item 92. The method of item 91, wherein the steps are repeated every 7 days. (Item 95) 1. A method for improving skin characteristics comprising: a. injecting a system comprising a first crosslinkable entity and a second crosslinkable entity; (i) each entity: (a) contains a cross-linkable moiety having a Log(P) in the range of 0 to 6; or (b) characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns; ii) the first crosslinkable entity and the second crosslinkable entity, when combined in vitro under physiological conditions, react with each other in a period of less than about 60 minutes at 37° C. (a) Storage modulus in the range of 50 Pa to 10 kPa; (b) The mass loss due to degradation over a period of 3 days under physiological conditions is less than 20%. forming a crosslinked material characterized by: (Item 96) 1. A method for improving skin characteristics comprising: a. injecting a system comprising a first crosslinkable entity and a second crosslinkable entity; (i) each entity: (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to intact skin; or (b) characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns; ii) the first crosslinkable entity and the second crosslinkable entity, when combined in vitro under physiological conditions, react with each other in a period of less than about 60 minutes at 37° C. (a) Storage modulus in the range of 50 Pa to 10 kPa; (b) The mass loss due to degradation over a period of 3 days under physiological conditions is less than 20%. forming a crosslinked material characterized by: (Item 97) 1. A method for improving skin characteristics comprising: a. injecting a system comprising a first crosslinkable entity and a second crosslinkable entity; (i) each entity: (a) is characterized by penetrating into and / or below the stratum corneum when applied ex vivo to intact skin; or (b) characterized by sufficient lipophilicity to achieve skin penetration of at least 50 microns; ii) the first crosslinkable entity and the second crosslinkable entity, when combined in vitro under physiological conditions, react with each other in a period of less than about 60 minutes at 37° C. (a) Storage modulus in the range of 50 Pa to 10 kPa; (b) the crosslinked material degrades at physiological conditions over a period of three days slower than the decomposition of said first and / or second crosslinkable entities in the absence of crosslinking. forming a crosslinked material characterized by: (Item 98) 98. The method of claim 97, wherein the system is injected using a double bore needle. (Item 99) 99. The method of claim 98, wherein the system is injected using a microneedle.

Claims

[Claim 1] The invention described in this specification.

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