Methods and systems for delivery of modified agarose polysaccharides

Abstract

Certain embodiments of the present disclosure generally relate to modified agarose polysaccharides for delivery using various techniques, such as injection. For example, some aspects are generally directed to modified agarose polysaccharides that are sterile and can be administered into the skin of a subject for various applications. In some cases, the modified agarose polysaccharides may have physical properties that allow them to be readily injected through a needle. Other aspects are generally directed to systems or methods for making or using such modified agarose polysaccharides, kits including such modified agarose polysaccharides, or the like.

Description

[0001] METHODS AND SYSTEMS FOR DELIVERY OF MODIFIED AGAROSE POLYSACCHARIDES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 578,479, filed August 24, 2023, entitled “Methods and Systems for Delivery of Modified Agarose Polysaccharides,” by Sarem, el al., incorporated herein by reference in its entirety.

[0004] FIELD

[0005] Certain embodiments of the present disclosure generally relate to modified agarose polysaccharides for delivery using various techniques, such as injection.

[0006] BACKGROUND

[0007] Hydrogel-forming polysaccharides such as agarose have had a tremendous impact on many areas of biomedical sciences. While historically their use was limited to the culture of bacteria, and electrophoresis, in recent years hydrogels have find applications as mammalian cell culture substrate and plant substrate. The application fields have been expanded to include the development of processing methods for the use of polysaccharide-based hydrogels as bioink (extrudable material that can encapsulate cells or serve as a carrier or substrate for cells), tissue filler, in vivo cell delivery, and drug delivery systems. In many of these applications, controlling the molecular weight of the polysaccharide is important to obtain precise mechanical properties of the hydrogel.

[0008] Polysaccharides are natural molecules which are synthesized by plants (cellulose, lignin), algae (agarose, alginate), insect (chitin), mammalian cells (hyaluronic acid), or certain genetically modified organisms. These polysaccharides can be synthesized in living organism by enzymes and similarly, can be depolymerized by enzymes. Among these natural polysaccharides, agarose, a polysaccharide that can be extracted from red seaweed, has been used for biomedical applications for over a century for applications such as growing prokaryotic cells, DNA electrophoresis and most recently for 3D cell culture assays.

[0009] However, due to the limited physico-mechanical properties of native agarose (NA), which can only be manipulated by changing the polymer concentration or the addition of other polysaccharides such as hyaluronic acid, chemical modifications of NA have been proposed which has led to a new class of polysaccharide so-called modified agarose polysaccharide (MAPs). See, e.g., Int. Pat. Apl. Pub. No. WO 2012 / 055596, published May 3, 2012; Int. Pat. Apl. Pub. No. WO 2013 / 023793 published February 21 , 2013; U.S. Pat. No. 9,388,252, issued July 12, 2016; or U.S. Pat. No. 10,968,285, issued April 6, 2021.

[0010] For certain clinical applications, however, MAP-containing hydrogels may need to be delivered in sterile form, and in a form such that they can be easily injected, e.g., through a needle, or through other techniques such as jet injectors. However, sterilizing hydrogels without impacting key properties such as rheology and injectability can be challenging especially, as the temperatures necessary to sterilize such hydrogels can also cause such hydrogels to break down chemically. In addition, many hydrogel formulations designed to withstand high temperatures may not be sufficiently flowable, and thus may be difficult to administer (for example, due to extensive crosslinking). Accordingly, improvements are needed.

[0011] SUMMARY

[0012] Certain embodiments of the present disclosure generally relate to modified agarose polysaccharides for delivery using various techniques, such as injection. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0013] One aspect is generally drawn to a composition. In accordance with one set of embodiments, the composition comprises a modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, an anesthetic, and a bioburden of no more than 100 CFU / ml.

[0014] Another set of embodiments is generally drawn to a composition, comprising a modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, an anesthetic, and an endotoxin concentration of less than 0.5 EU / ml.

[0015] The composition, in yet another set of embodiments, comprises a modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, an anesthetic, and an endotoxin concentration of less than 20 EU / ml.

[0016] Another aspect is generally drawn to a device. According to one set of embodiments, the device comprises a syringe comprising a barrel. The barrel may contain a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa. In some cases, the composition exhibits a bioburden of no more than 100 CFU / ml.

[0017] The device, in another set of embodiments, comprises a syringe comprising a barrel. In some cases, the barrel contains a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa. In some embodiments, the modified agarose polysaccharide has an endotoxin concentration of less than 0.5 EU / ml.

[0018] In yet another set of embodiments, the device comprises a syringe comprising a barrel. In some embodiments, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa. In certain cases, the modified agarose polysaccharide has an endotoxin concentration of less than 20 EU / ml.

[0019] In another set of embodiments, the device comprises a syringe comprising a barrel. In some cases, the barrel may contain autoclaved modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa.

[0020] The device, in yet another set of embodiments, comprises a syringe comprising a barrel and a needle. In certain embodiments, the barrel contains heat-treated modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa.

[0021] Still another aspect is generally drawn to a method. In accordance with one set of embodiments, the method comprises injecting, into a subject, a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa. In certain cases, the composition exhibits a bioburden of no more 100 CFU / ml.

[0022] The method, in another set of embodiments, comprises injecting, into a subject, a comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 0.5 EU / ml.

[0023] In yet another set of embodiments, the method comprises injecting, into a subject, a comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 20 EU / ml.

[0024] Yet another aspect is generally drawn to a kit. In one set of embodiments, the kit comprises a syringe comprising a barrel. In certain embodiments, the barrel contains a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa. In certain cases, the modified agarose polysaccharide exhibits a bioburden of no more 100 CFU / ml.

[0025] In another set of embodiments, the kit comprises a syringe comprising a barrel, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 0.5 EU / ml. In yet another set of embodiments, the kit comprises a syringe comprising a barrel, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 20 EU / ml.

[0026] Another aspect is generally drawn to the use of a composition for the treatment of a subject. In one set of embodiments, the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa. In certain cases, the modified agarose polysaccharide exhibits a bioburden of no more than 100 CFU / ml.

[0027] In another set of embodiments, the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 0.5 EU / ml.

[0028] The composition, in still another set of embodiments, comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 20 EU / ml.

[0029] Still another aspect is generally drawn to the use of a composition for the manufacture of a medicament for the treatment of a medical condition in a subject. In one set of embodiments, the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa. In certain cases, the modified agarose polysaccharide exhibits a bioburden of no more than 100 CFU / ml.

[0030] The composition, in another set of embodiments, comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 0.5 EU / ml.

[0031] In yet another set of embodiments, the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 20 EU / ml.

[0032] Several methods are disclosed herein of administering a subject with a composition for prevention or treatment of a particular condition. It is to be understood that in each such aspect of the disclosure, the disclosure specifically includes, also, the composition for use in the treatment or prevention of that particular condition, as well as use of the composition for the manufacture of a medicament for the treatment or prevention of that particular condition.

[0033] In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, a composition comprising a modified agarose polysaccharide. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, a composition comprising a modified agarose polysaccharide.

[0034] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0037] Fig. 1 illustrates a method of producing modified agarose polysaccharides, according to one embodiment; and

[0038] Fig. 2 illustrates data showing certain modified agarose polysaccharides retain their shear moduli after autoclaving, in another embodiment.

[0039] DETAILED DESCRIPTION

[0040] Certain embodiments of the present disclosure generally relate to modified agarose polysaccharides for delivery using various techniques, such as injection. For example, some aspects are generally directed to modified agarose polysaccharides that are sterile and can be administered into the skin of a subject for various applications. In some cases, the modified agarose polysaccharides may have physical properties that allow them to be readily injected through a needle. Other aspects are generally directed to systems or methods for making or using such modified agarose polysaccharides, kits including such modified agarose polysaccharides, or the like.

[0041] For example, certain aspects are generally drawn to modified agarose polysaccharides that have been prepared or treated such that they can flow through a needle, for example for administration to a subject. Modified agarose polysaccharides that are too viscous may be difficult to force through a needle into a subject, e.g., by medical personnel attempting to administer the modified agarose polysaccharide to a subject. Accordingly, such modified agarose polysaccharides have to be delivered using other techniques (e.g., manual placement), which can make it difficult to administer the modified agarose polysaccharides precisely in a subject, e.g., in the skin of a subject. Accordingly, as discussed herein, certain embodiments are generally directed to modified agarose polysaccharides that can be readily administered to a subject, e.g., by injection through a needle.

[0042] Another difficulty is that such modified agarose polysaccharides should be administered in a relatively sterile formulation, e.g., if they are to be administered to a subject, such as injected into the skin. While modified agarose polysaccharides can be autoclaved or treated at relatively high temperatures (e.g., above 100 °C), such treatments may also degrade the modified agarose polysaccharides, rendering it difficult to control its viscosity after autoclaving. Furthermore, because of the need to maintain sterility and their relatively high viscosities, it can be difficult to sterilize the modified agarose polysaccharides before introducing them into a barrel of a syringe, while it can also be difficult to sterilize the modified agarose polysaccharides after introducing them into the barrel of a syringe, as the modified agarose polysaccharide may break down under creatin conditions during sterilization.

[0043] Accordingly, certain embodiments such as discussed herein are generally directed to modified agarose polysaccharides that can be sterilized, e.g., within the barrel of a syringe. In some cases, the modified agarose polysaccharide may be prepared using pH buffers that are able to control the pH of the agarose, e.g., during formation and / or during sterilization. Without wishing to be bound by any theory, it is believed that the formation of H+ions during heatsterilization may be a factor in causing degradation to occur, and thus, by controlling the pH of the modified agarose polysaccharide, the amount of degradation of the modified agarose polysaccharide can be controlled or minimized.

[0044] For instance, one set of embodiments may be directed to a barrel or other container containing modified agarose polysaccharide that is relatively flowable and is substantively sterile. For example, the modified agarose polysaccharide may have a shear modulus G’ of between 10 Pa and 20 kPa. The sterility of the modified agarose polysaccharide may be determined, for example, by determining a bioburden of no more than 100 CFU / ml, and / or an endotoxin concentration of less than 20 EU / ml or less than 0.5 EU / ml. Other examples of such modified agarose polysaccharides are discussed in more detail below. In some cases, the modified agarose polysaccharides may have a structure that is based on agarose, but that some of the agarose subunits contain moictics such as carboxylate, halogen, sulfate, sulfonate, phosphorylate, etc., rather than hydroxides.

[0045] Such modified agarose polysaccharides can be used for a variety of applications, such as those discussed herein. For example, such modified agarose polysaccharides can be used for improving the appearance of skin, preventing and / or treating hair loss, filling wrinkles or contouring the face or body of a subject.

[0046] The above discussion is a non-limiting example of one embodiment of the present disclosure that can be used to produce modified agarose polysaccharides for injection and other applications. However, other embodiments are also possible. Accordingly, more generally, various aspects are directed to modified agarose polysaccharides for various applications.

[0047] For example, certain aspects such as described herein are generally directed to modified agarose polysaccharides. Agaroses can be found in several sources including agar which is a structural polysaccharide of the cell walls of a variety of red algae. Important sources of agar are Gelidiaceae such as Gelidium amansii, Gelidium japonicum, Gelidium pacificum, Gelidium subcostatum, Pterocladia tenuis and Acanthopeltis japonica, red algae belonging to Gracilariaceae such as Gracilaria verrucosa and Gracilaria gigas, red algae belonging to Ceramiaceae such as Ceramium kondoi and Campylaephora hypnaeoides . Agar includes two groups of polysaccharides, agarose and agaropectin. Agarose is a neutral, linear polysaccharide with no branching and has a backbone of 1,3-linked beta-D-galactose-(l-4)-alpha-L-3,6 anhydrogalactose repeat units.

[0048] Such dimeric repeat units derived from naturally occurring polysaccharides can be chemically modified to produce a variety of modified agarose polysaccharides, including those describe herein. Such dimeric repeating units may be derived from naturally occurring polysaccharides, and in some cases may be chemically modified by the specific oxidation of the primary hydroxyl, for example, to form carboxylate moieties, halogenated moieties, sulfate moieties, sulfonated moieties, phosphorylated moieties, or the like.

[0049] In one embodiment, the modified agarose polysaccharide contains at least one modified polysaccharide. In some cases, the modified agarose polysaccharide may have a structure: wherein the R’s may each be independently of any of a variety of groups, such as carboxylate, halogen, sulfate, sulfonate, and phosphorylate. Examples of halogens include chlorine, fluorine, bromine, iodine, etc. In unmodified agarose, the R’s are hydroxides. However, in a modified agarose polysaccharide, at least some of the R moictics arc not hydroxides. For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or substantially all of the R moieties in the above structure are not hydroxides (e.g., on a mole basis). Instead, the R’s may be moieties such as carboxylate, halogen, sulfate, sulfonate, phosphorylate, etc., and may be present in any suitable arrangement in the modified agarose polysaccharide. One or more than one such moiety may be present, e.g., a modified agarose polysaccharide may contain only carboxylate, only carboxylate and hydroxide, carboxylate and halogen, halogen and hydroxide, carboxylate and halogen and hydroxide, etc. Such moieties may appear randomly throughout the structure, or in a systematic or repeating arrangement.

[0050] In addition, in some cases, no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of the R moieties are not hydroxides. Combinations of any of these ranges are also possible in certain cases; for example, in a modified agarose polysaccharide, between 10% and 95%, between 20% and 90%, between 50% and 95%, etc. of the R moieties in the above structure may not be hydroxides, but may be other moictics such as carboxylate, halogen, sulfate, sulfonate, phosphorylate, etc.

[0051] There may be only one type of oxidized moiety present on the modified agarose polysaccharide, or there may be 2, 3, or more types of oxidized moieties present on the modified agarose polysaccharide. As non-limiting examples, all of the oxidized moieties may be carboxylates or carboxylic acids, the oxidized moieties may all be phosphorylated, or there may be a mixture of two or more moieties, such as phosphorylates and sulfonates, etc.

[0052] In some embodiments, at least 5% of the modified agarose polysaccharide contains oxidized moieties, relative to native agarose (e.g., such that at least 5% of the hydroxyls on the disaccharide units are other, oxidized moieties), and in some cases, at least 10%, at least 11%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or substantially all of the modified agarose polysaccharide contains oxidized moieties instead of hydroxyls. Non-limiting examples of oxidized moieties include carboxylates, sulfates, sulfonates, phosphorylates, etc. In some cases, no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the modified agarose polysaccharide contains oxidized moieties, relative to native agarose. Combinations of any of the following are also possible. As non-limiting examples, at least 20% to 99%, at least 50% to 95%, at least 60% to 80%, etc. of the modified agarose polysaccharide may contain oxidized moieties. These are typically determined as a percentage by number or a mole basis. Percentages such as these can be achieved, for example, by oxidizing native agarose in a controlled manner, or in some cases, oxidizing it completely so that about 100% of the hydroxyls have been oxidized.

[0053] A variety of techniques can be used to prepare a modified agarose polysaccharide, e.g., having the above structure. For example, in one embodiment, an agarose can be reacted to oxidize at least some of the alcohol groups within the structure to form a carboxylic acid. Such oxidation can be carried out, as a non-limiting example, by sodium hypochlorite in the presence of 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO). The reaction mechanism is shown in Fig. 1. Other oxidation reactions, e.g., of the alcohol groups within the structure, are also known to those of ordinary skill in the ail. In addition, in some cases, the oxidation of the alcohol groups can be carried out by an enzymatic process or using a bacteriological system, etc.

[0054] The modified agarose polysaccharides may be present in a range of sizes and / or molecular weights. In some cases, the molecular weight of the modified agarose polysaccharide may be at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 21 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa, at least 50 kDa, at least 55 kDa, at least 60 kDa, at least 65 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 85 kDa, at least 90 kDa, at least 100 kDa, at least 105 kDa, at least 110 kDa, at least 115 kDa, at least 120 kDa, at least 125 kDa, at least 130 kDa, at least 135 kDa, at least 140 kDa, at least 145 kDa, at least 150 kDa, at least 155 kDa, at least 160 kDa, at least 165 kDa, at least 170 kDa, at least 175 kDa, at least 180 kDa, at least 185 kDa, at least 190 kDa, at least 195 kDa, at least 200 kDa, at least 210 kDa, at least 220 kDa, at least 230 kDa, at least 240 kDa, at least 250 kDa, etc. In addition in some cases, the molecular weight of the modified agarose polysaccharide may be no more than 250 kDa, no more than 240 kDa, no more than 230 kDa, no more than 220 kDa, no more than 210 kDa, no more than 200 kDa, no more than 195 kDa, no more than 190 kDa, no more than 185 kDa, no more than 180 kDa, no more than 175 kDa, no more than 170 kDa, no more than 165 kDa, no more than 160 kDa, no more than 155 kDa, no more than 150 kDa, no more than 145 kDa, no more than 140 kDa, no more than 135 kDa, no more than 130 kDa, no more than 125 kDa, no more than 120 kDa, no more than 115 kDa, no more than 110 kDa, no more than 105 kDa, no more than 100 kDa, no more than 95 kDa, no more than 90 kDa, no more than 85 kDa, no more than 80 kDa, no more than 75 kDa, no more than 70 kDa, no more than 65 kDa, no more than 60 kDa, no more than 55 kDa, no more than 50 kDa, no more than 45 kDa, no more than 40 kDa, no more than 35 kDa, no more than 30 kDa, no more than 25 kDa, no more than 20 kDa, no more than 15 kDa, no more than 10 kDa, etc. Molecular weights of the modified agarose polysaccharides may be determined on a weightaverage basis. Combinations of the above ranges are also possible, e.g., the modified agarose polysaccharide may have a weight average molecular weight of between 21 kDa and 180 kDa, between 10 kDa and 150 kDa, between 90 kDa and 130 kDa, between 45 kDa and 85 kDa, etc.

[0055] In some cases, the modified agarose polysaccharide may have relatively low poly dispersity. For instance, the polydispersity of the modified agarose polysaccharide may be at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.8, at least 2. In some cases, the polydispersity of the modified agarose polysaccharide may be no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.8, no more than 2.6, no more than 2.4, no more than 2.2, no more than 2.0, etc. Combinations of any of these are also possible. For example, the modified agarose polysaccharide may have a polydispersity index of between 1 .5 and 3.2, between 1 .6 and 2.6, between 1 and 3, between 1.8 and 2.4, or the like.

[0056] The modified agarose polysaccharide may, in certain embodiments, exhibit an at least partial 0-sheet structure. The modified agarose polysaccharide may adopt an at least partial P- sheet structure due to the presence of oxidized moieties, relative to native agarose. The modified agarose polysaccharide may exhibit, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% P-sheet structure. The P-sheet structure can be determined, e.g., by circular dichroism, Raman optical spectroscopy, or by other suitable techniques known to those of ordinary skill in the art. However, it should not be assumed that this percentage is necessarily the same as the percentage of oxidized moieties within the modified agarose polysaccharide (although it can be).

[0057] In some embodiments, one or more side chains of the modified agarose polysaccharide may include a peptide or protein sequence. Non-limiting examples include the cell adhesion sequence arginine-glycine-aspartic acid (RGD), the peptide sequences IKVAV (SEQ ID NO: 1) and YIGSR (SEQ ID NO: 2) or a protein such as collagen, collagen fragments, fibronectin, hyaluronic acid, etc. In yet other embodiments, one or more side chains of the modified agarose polysaccharide may include a nucleic acid sequence. The nucleic acid sequence may comprise single- stranded DNA, double- stranded DNA, single-stranded RNA, siRNA, etc.

[0058] In addition, it should be understood that in some cases, different modified agarose polysaccharides may be present, and / or the modified agarose polysaccharides may be mixed together with native agaroses, in various embodiments. For example, a first modified agarose polysaccharide containing a first type of oxidized moiety may be mixed or blended together with a second modified agarose polysaccharide containing a second type of oxidized moiety, and / or with another first modified agarose polysaccharide containing a first type of oxidized moiety having a different molecular' weight, and / or with native agarose, etc. Thus, there are a variety of different types of formulations, containing different amounts or concentrations of various modified agarose polysaccharides, that can be used.

[0059] In some aspects, the modified agarose polysaccharide may be present in a formulation. For instance, the modified agarose polysaccharide may be present in a gel, such as a hydrogel. In addition, in certain embodiments, more than one such modified agarose polysaccharide may be present in a formulation. For example, in one set of embodiments, a formulation may contain two, three, or more modified agarose polysaccharides, e.g., as discussed herein. In some embodiments, other polymers may be present within a formulation, for example, hyaluronic acid, poly-L-lactic acid, poly(methylmethacrylate), calcium salts (including, for example, calcium phosphate, calcium carbonate, etc.) another modified agarose polysaccharide including any of those discussed herein, etc.

[0060] In one set of embodiments, the formulation may have a shear modulus that can range, for example, from about 10 Pa (e.g., which reflects the structure of a nerve tissue) to about 107Pa (e.g., which corresponds with the shear modulus of cartilage tissues). By using agaroses and modified agarose polysaccharides with different properties, chemical modifications, etc., the structure of the formulation can be controlled, for example, to have a variety of shear moduli, depending on the particular application.

[0061] For example, the formulation containing a modified agarose polysaccharide such as discussed herein may have a shear modulus of at least 1 Pa, at least 3 Pa, at last 5 Pa, least 10 Pa, at least 20 Pa, at least 30 Pa, at least 50 Pa, at least 100 Pa, at least 300 Pa, at least 500 Pa, at least 1 kPa, at least 3 kPa, at least 5 kPa, at least 10 kPa, at least 30 kPa, at least 50 kPa, at least 100 kPa, etc. In addition, the formulation may have a shear modulus of no more than 10 MPa, no more than 5 MPa, no more than 3 MPa, no more than 1 MPa, no more than 500 kPa, no more than 300 kPa, no more than 100 kPa, no more than 50 kPa, no more than 30 kPa, no more than 10 kPa, no more than 5 kPa, no more than 3 kPa, no more than 1 kPa, no more than 500 Pa, no more than 300 Pa, no more than 100 Pa, no more than 50 Pa, no more than 30 Pa, no more than 10 Pa, no more than 5 Pa, no more than 3 Pa, etc. Combinations of any of these are possible. For example, the shear modulus of a formulation may be between 1 Pa and 100 kPa, between 1 Pa and 50 kPa, between 10 Pa and 10 kPa, between 100 kPa and 5 MPa, etc.

[0062] In addition, in one set of embodiments, the formulation containing a modified agarose polysaccharide such as discussed herein may have any suitable average molecular weight of the modified agarose polysaccharides within the formulation. By using agaroses and modified agarose polysaccharides with different properties, chemical modifications, etc., the overall molecular weight of the modified agarose polysaccharides within the formulation can be controlled. For example, the average molecular weight of the modified agarose polysaccharides may be at least at least 5 kDa, at least 10 kDa, at least 30 kDa, at least 50 kDa, at least 100 kDa, at least 300 kDa, at least 500 kDa, at least 1 MDa, etc. In addition, the molecular weight may be no more than 1 MDa, no more than 500 kDa, no more than 300 kDa, no more than 100 kDa, no more than 50 kDa, no more than 30 kDa, no more than 10 kDa, no more than 5 kDa, etc. Combinations of any of these arc possible. For example, the average molecular weight of the modified agarose polysaccharides within a formulation may be between 1 Da and 100 kDa, between 1 Da to 50 kDa, between 10 Da to 10 kDa, etc.

[0063] In some embodiments, other materials may also be present in the formulation, e.g., in addition to one or more modified agarose polysaccharides. For example, the formulation may include a solute such as saline, or other components such hyaluronic acid, calcium hydroxylapatite, poly-L-lactic acid, poly(methylmethacrylate), heparin sulfate, dermatan sulfate, chondroitin sulfate, alginate, chitosan, pullulan, k-carrageenan, silk, minerals, vitamins, proteins, peptides, or the like. As still other examples, pharmaceutically active agents such as growth factors, insulin, biologically active peptides, chemokines, cytokines, steroids, antibiotics, analgesics, anti-inflammatory agents, anti-cancer drugs, local anesthetics such as lidocaine, amino acids, antioxidants, vitamins, minerals, neurotoxic proteins such as Botulinum toxin or the like may be present within a formulation. In addition, in some embodiments, the formulation may be used for diagnostic purposes. For example, the formulation may include imaging agents such as MRI contrast agents, CT contrast agents, fluorescent imaging probes, radioactive nuclei, etc.

[0064] For example, in one aspect, the formulation may contain an anesthetic. As discussed herein, the formulation may be one that is to be injected or administered to the skin of a subject. In some case, the formulation may be preset within a barrel of a syringe, e.g., to be injected via a needle into the subject. However, many gels are often viscous and difficult to administer through a needle, e.g., for applications where an anesthetic may be useful. Furthermore, it is difficult to sterilize such gels within the barrel of a syringe, e.g., without adversely affecting its chemical properties (for example, resulting in degradation of the gel, adverse chemical reactions, or the like). Accordingly, without wishing to be bound by any theory, it is believed that certain formulations containing modified agarose polysaccharides and anesthetics that can be readily administered through a needle, such as are described herein, have not previously been achievable, due to the difficulty in making such formulations both sterile and injectable through a needle.

[0065] Non-limiting examples of anesthetics lignocaine (lidocaine), bupivacaine, butanilicaine, carticaine, cinchocaine (dibucaine), clibucaine, ethylparapiperidinoacetylaminobenzoate, etidocaine, mepivacaine, oxethazaine, prilocaine,ropivacaine, tolycaine, trimecaine, vadocaine, articaine, levobupivacaine, amylocaine, cocaine, propanocaine,clormecaine, cyclomethycaine, proxymetacaine, amethocaine (tetracaine), benzocaine, butacaine, butoxycaine, butyl aminobenzoate, chloroprocaine, dimethocaine (larocaine), oxybuprocaine, piperocaine, parethoxycaine, procaine (novocaine), propoxycaine, tricaine, etc. Combinations of any one, two, or more of these, and / or other anesthetics, can also be used in certain instances.

[0066] The anesthetic may be present at any suitable amount or concentration within the formulation. For example, the anesthetic may be present at a concentration of at least 1 mM, at least 2 mM, at least 3 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 50 mM, at least 100 mM, and / or no more than 100 mM, no more than 50 mM, no more than 30 mM, no more than 20 mM, no more than 10 mM, no more than 5 mM, no more than 3 mM, no more than 2 mM, no more than 1 mM, etc. In some embodiments, the anesthetic may include lidocaine.

[0067] In one aspect, the formulation may be present in a device for injection into a subject, e.g., into the skin of a subject. For example, the formulation may be present in a barrel of a syringe. The barrel of the syringe, in some embodiments, may be formed from a medically acceptable material. For example, the barrel may include materials such as glass or polymers (e.g., a cyclic olefin copolymer). The barrel may be attached to a needle, a cannula, or other suitable device for injection or administration to a subject. In some cases, the needle may have an inner diameter of 1 mm or less, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, 0.6 mm or less, 0.5 mm or less, 0.4 mm or less, or 0.3 mm or less.

[0068] In one aspect, the modified agarose polysaccharide may be one that has been sterilized, e.g., heat-treated to render the modified agarose polysaccharide sterile. For example, the modified agarose polysaccharide may be exposed to a temperature of at least 90 °C, at least 100 °C, at least 105 °C, at least 110 °C, at least 115 °C, at least 120 °C, at least 125 °C, at least 130 °C, at least 135 °C, at least 140 °C, at least 145 °C, etc. In some cases, the modified agarose polysaccharide may be exposed to such temperatures for a period of time of at least at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes at, least 5 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, or at least 1 hour. In one set of embodiments, the modified agarose polysaccharide may be autoclaved to render it sterile.

[0069] The sterility of the composition may be determined, in certain cases, by measuring the bioburden of the resulting composition. In some cases, the sterility bioburden may be determined before and / or after sterilization. The bioburden may be determined as the number of bacteria or other microorganisms present within the composition. In some cases, the bioburden may be determined as a quantification of colony forming units (CFU), e.g., the number of colonies or cultures that an agar plate grows after exposure to the composition. The number of visible colonies can be determined on the agar plate and can be measured, e.g., as CFU / ml. Standardized methods for determining bioburdens are known to those of ordinary skill in the art.

[0070] In one set of embodiments, the composition may have a bioburden of no more than 1000 CFU / ml, no more than 500 CFU / ml, no more than 300 CFU / ml, no more than 200 CFU / ml, no more than 100 CFU / ml, no more than 50 CFU / ml, no more than 30 CFU / ml, no more than 20 CFU / ml, no more than 10 CFU / ml, no more than 5 CFU / ml, no more than 3 CFU / ml, no more than 1 CFU / ml, etc. As discussed herein, it is difficult to sterilize a modified agarose polysaccharide without substantial degradation or deterioration of properties, and thus, most modified agarose polysaccharides are not sterilized, e.g., to such bioburden levels.

[0071] In another set of embodiments, the sterility of the composition may be determined by measuring an endotoxin concentration. Endotoxin concentrations are typically determined as EU (endotoxin units) per milliliter. The endotoxin concentration may be less than 1000 EU / ml, less than 500 EU / ml, less than 300 EU / ml, less than 100 EU / ml, less than 80 EU / ml, less than 80 EU / ml, less than 60 EU / ml, less than 40 EU / ml, than 20 EU / ml, less than 10 EU / ml, less than 5 EU / ml, less than 3 EU / ml, less than 1 EU / ml, less than 0.5 EU / ml, less than 0.3 EU / ml, less than 0.1 EU / ml, less than 0.05 EU / ml, less than 0.03 EU / ml, less than 0.01 EU / ml, etc. Those of ordinary skill in the art will be aware of methods for determining endotoxins in a sample, e.g., in a pharmaceutical product or medical device or a combination device.

[0072] In addition, certain aspects are generally directed to compositions that, when sterilized, do not substantially exhibit degradation. In some embodiments, the amount of degradation can be determined as a change in shear modulus, before the composition is sterilized as compared to after the composition is sterilized. For example, the composition may exhibit a change (e.g., a loss) of shear modulus of no more than 30%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of the shear modulus after sterilization, relative to the shear modulus before sterilization.

[0073] Without wishing to be bound by any theory, it is believed that under certain conditions, heat applied to the formulation may cause a substantial change in pH, which can cause the degradation of the modified agarose polysaccharide, e.g., as determinable by a significant change in shear modulus, or other physical properties. Accordingly, in certain embodiments, a pH buffer may be present to the composition, e.g., to reduce the change in pH, and thus lessen the amount of degradation that may occur during sterilization. The pH buffer may be phosphate- buffered saline, or other types of pH buffer, e.g., saline or Ringer’s solution.

[0074] In addition, in accordance with certain embodiments, the pH buffer may be one that has relatively low concentrations of potassium, or is substantially free of potassium in certain cases. For example, the concentration of potassium in the pH buffer, or in the composition including the pH buffer, may be no more than 10 m ill i molar, no more than 5 millimolar, no more than 3 millimolar, no more than 1 millimolar, no more than 0.5 millimolar, no more than 0.3 millimolar, no more than 0.2 millimolar, or no more than 0.1 millimolar, etc.

[0075] In some cases, the pH value can be at least 7, at least 7.1, at least 7.2, at least 7.3, at least 7.4, at least 7.5, at least 7.6, at least 7.7, at least 7.8, at least 7.9, and / or no more than 8, no more than 7.9, no more than 7.8, no more than 7.7, no more than 7.6, no more than 7.5, no more than 7.4, no more than 7.3, no more than 7.2, no more than 7.1, etc. Combinations of these are also possible, e.g., the pH may be between 7 and 8, between 7.2 and 7.4, between 7 and 7.6, between 7.5 and 7.9, between 7.4 and 7.8, between 7.3 and 7.5, etc.

[0076] In some cases, the osmolarity may be at least 250 mOsm / kg, at least 260 mOsm / kg, at least 270 mOsm / kg, at least 280 mOsm / kg, at least 290 mOsm / kg, at least 300 mOsm / kg, at least 310 mOsm / kg, at least 320 mOsm / kg, at least 330 mOsm / kg, at least 340 mOsm / kg, at least 350 mOsm / kg, at least 360 mOsm / kg, at least 370 mOsm / kg, and / or no more than 400 mOsm / kg, no more than 390 mOsm / kg, no more than 380 mOsm / kg, no more than 370 mOsm / kg, no more than 360 mOsm / kg, no more than 350 mOsm / kg, no more than 340 mOsm / kg, no more than 330 mOsm / kg, no more than 320 mOsm / kg, no more than 310 mOsm / kg, no more than 300 mOsm / kg, no more than 290 mOsm / kg, no more than 280 mOsm / kg, etc. Combinations of these are also possible, e.g., the osmolarity may be between 300 mOsm / kg and 350 mOsm / kg, between 290 mOsm / kg and 310 mOsm / kg, between 270 and 330 mOsm / kg, between 310 mOsm / kg and 400 mOsm / kg, between 340 mOsm / kg and 370 mOsm / kg, etc.

[0077] In addition, certain aspects are generally directed to applications or uses of compositions or devices such as those described herein. For example, in one set of embodiments, a composition may be used to improve the appearance of the skin of a subject, to treat the wound of a subject, or other uses such as those described herein. For instance, a composition may be administered as described herein, e.g., into or under the skin of a subject, such as a human or non-human subject. For instance, the composition can be injected into the cutis and / or the subcutis of a subject. The composition may be administered to any suitable location, e.g., to the face or body of a subject. In some cases, the composition may be administered to a particular location, e.g., to a wound of the subject, to a wrinkle in the subject, or the like.

[0078] Examples of non-human subjects include, but are not limited to, a mammal such as a cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, cat, a primate (e.g., a monkey, a chimpanzee, etc.), or the like. In some cases, improved skin appearance can be determined using different scoring systems that are established and used frequently in clinical trials, such as the Wrinkle Assessment Score. The Wrinkle Assessment Score is a system used to objectively evaluate the severity of wrinkles on a patient's skin. It is a numeric score that is assigned based on the appearance of wrinkles on the skin, with higher scores indicating more severe wrinkles. The Wrinkle Assessment Score is typically calculated using a standardized set of criteria, such as the number and depth of wrinkles, the location of the wrinkles, and the overall appearance of the skin. A dermatologist may also take into account other factors that can contribute to wrinkles, such as sun damage, smoking, and genetics.

[0079] In some cases, for instance, the composition may be injected into the skin of a subject to fill wrinkles, and / or to contour the subject’s skin, e.g., the face or body of the subject. Wrinkles may include lines or creases that form in the skin due to various factors such as aging, repetitive, facial expressions, sun damage, smoking, and environmental factors. Wrinkles are generally a natural pail of the aging process and can appear on any part of the body, but are most commonly seen on the face, neck, and hands. They may occur when the skin's collagen and elastin fibers begin to break down, leading to a loss of skin elasticity and firmness. Wrinkles may include fine lines and deep wrinkles. Fine lines are shallow and often appear around the eyes and mouth, while deep wrinkles are more pronounced and can appear on the forehead and cheeks. In various embodiments, the composition may be administered to a subject to treat a condition of the subject, such as for wound healing, treatment of dry skin conditions and sundamaged skin, treatment of hyperpigmentation disorders, treatment and / or prevention of hair loss, treatment of conditions that have inflammation as a component of the disease process, such as psoriasis (e.g., asteototic eczema), etc.

[0080] For example, sun damage in a subject may be determined visually, e.g., based on the appearance of brown spots, uneven skin tone, fine wrinkles, irregular moles or lesions, or the like. In some cases, cameras that can take UV photographs of the skin can reveal areas of sun damage that may not be visible to the naked eye. For example, these photographs can show areas of hyperpigmentation, fine lines, and other signs of sun damage. In addition, a biopsy may be used to examine the skin for signs of sun damage. In some cases, a Wood's lamp, which emits black or ultraviolet light, can be used to examine the skin for signs of sun damage. This can help reveal areas of hyperpigmentation that may not be visible under normal light. In some cases, repeated exposed to ultraviolet light, e.g., from the sun, may cause dermal cells to both decrease their production of hyaluronan as well as increase their rate of degradation. This loss of material may result in various skin conditions such as, e.g., wrinkling, hollowness, loss of moisture and other undesirable conditions that contribute to the appearance of aging. In certain embodiments, compositions such as those described herein can be administered to the skin of a subject to replace lost endogenous matrix polymers, or enhance / facilitate the function of existing matrix polymers, in order to treat these skin conditions.

[0081] As another example, in certain cases, the composition may be applied to dry skin in a subject having dry skin. Such dry skin can be determined in a subject, for example, by using a comeometer to measure hydration levels in the skin. A corneometer measures the electrical capacitance of the skin, which is directly related to the amount of water in the skin, e.g., a lower reading on the corneometer indicates drier skin. For example, a value of less than 30 units may indicate dry skin. In addition, in some cases, dry skin can be determined using a variety of tests, such as the transepidermal water loss (TEWL) test. This test measures the amount of water that evaporates through the skin over a period of time. Higher TEWL values may indicate a weaker skin barrier, which can lead to dryness. In one set of embodiments, the skin may exhibit a TEWL value of 10 g / m2 / h, or higher. Dry skin can also be determined visually, e.g., be identifying flaky skin, skin irritation or inflammation, or the like. In another set of embodiments, the composition may be administered to the skin of a subject to prevent hair loss. For example, the composition may include components such as biomimetic peptides, enzymes, growth factors, minerals, vitamins, minoxidil, other hair treatment medications, or the like. In some embodiments, such components may promote blood circulation and / or rejuvenating hair follicles.

[0082] In yet another set of embodiments, the composition may be used as an implant, such as but not limited to a regenerative implant. For example, the composition may include cells, e.g., that can grow to form tissue. Non-limiting examples of regenerative implants include artificial skin, artificial blood vessels, artificial nerve tissues, or the like. Other examples include mucosal tissues or parts of the eye, e.g., artificial lenses. As another example, the composition can be used as a plastic surgery implant, e.g., for reconstructive and / or cosmetic surgery. For example, the composition may be implanted in a facial region such as a nose, a forehead, a jaw, a cheek. Other regions of the body include a breast, a hip, a calf, or the like.

[0083] In still another set of embodiments, the composition may be used as a controlled release formulation, e.g., of a drug or pharmaceutically active compound. In some cases, the composition may be implanted and allowed to release the drug or pharmaceutically active compound, i.e., as an implant, such as a regenerative implant. However, in other cases, the composition may be used only as a controlled release formulation, e.g., without necessarily being also used as an implant such as described herein. In some cases, the composition may be controlled, e.g., as discussed herein, to control the degree at which the drug or pharmaceutically active compound. Non-limiting examples of drugs or pharmaceutically active compound include anesthetics or other compounds such as those described herein. Other examples include, but are not limited to, growth factors, insulin, biologically active peptides, chemokines, cytokines, steroids, antibiotics, analgesics, anti-inflammatory agents, anti-cancer drugs, or the like. In some cases, more than one such compound may be present within a composition.

[0084] The composition, in accordance with yet another set of embodiments, may be used as a diagnostic. For example, the composition may include imaging agents such as magnetic resonance imaging (MRI) contrast agents, computed tomography (CT) contrast agents, fluorescent imaging probes radionuclei, or the like.

[0085] In certain aspects, compositions or devices such as those described herein may be contained in a kit. The kit may include a package or an assembly including one or more of the compositions described herein. Each of the compositions of the kit may be provided in any suitable form, c.g., in gel form, in liquid form (c.g., in solution), in solid form (c.g., a dried powder), etc. In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species, which may or may not be provided with the kit. Examples of other compositions or components that may be present include, but are not limited to, solvents, surfactants, diluents, salts, buffers, emulsifiers, chelating agents, fillers, antioxidants, binding agents, bulking agents, preservatives, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, dishes, frits, filters, rings, clamps, wraps, patches, containers, and the like, for example, for using, administering, modifying, assembling, storing, packaging, preparing, mixing, diluting, and / or preserving the compositions components for a particular use, for example, to a sample and / or a subject.

[0086] A kit may, in some cases, include instructions in any form that are provided in connection with the compositions or devices in such a manner that one of ordinary skill in the art would recognize that the instructions are to be associated with the compositions or devices. For instance, the instructions may include instructions for the use, modification, mixing, diluting, preserving, administering, assembly, storage, packaging, and / or preparation of the compositions or devices. In some cases, the instructions may also include instructions for the delivery and / or administration of the compositions, for example, for a particular use, e.g., to a sample and / or a subject. Examples for administration include any of those described herein. The instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions, for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications), provided in any manner.

[0087] U.S. Provisional Patent Application Serial No. 63 / 578,479, filed August 24, 2023, entitled “Methods and Systems for Delivery of Modified Agarose Polysaccharides,” by Sarem, et al., is incorporated herein by reference in its entirety.

[0088] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0089] EXAMPLE 1 A composition comprising modified agarose polysaccharide and lidocaine was prepared as described herein. A syringe was filed with the composition and then autoclaved different parameters commonly used in industry (e.g., heating to 128 °C for 3 min or 121 °C for 15 min). This was compared to a composition as described in the prior art, which was also used to fill a syringe and autoclaved using parameters commonly used in industry (e.g., heating to 127 °C for 4 min). The shear modulus G’ of each composition was determined before and after autoclaving (sterilization). This was determined using a rheometer with 0.1% strain, 0.1-10 Hz frequency sweep at 25 °C.

[0090] These results are shown in Fig. 2, with the prior art formulation in the dark bars on the left and the test formulations described above in light bars on the right. The shear moduli before and after sterilization are shown here. As can be seen, the prior art compositions showed significant degradation in shear modulus, exhibiting a loss of more than 50%. In contrast, the formulations as described herein surprisingly maintained shear modulus even after autoclaving.

[0091] EXAMPLE 2

[0092] This example illustrates certain buffers used in the prior art, and buffers that can be used to prepare compositions, e.g., as discussed herein.

[0093] Prior Art Buffer #1 :

[0094] Prior Art Buffer #2:

[0095] The pH buffer may be one that has relatively low concentrations of potassium, or is substantially free of potassium in certain cases. In some cases, the pH value could range from 7 to 7.6, while the osmolarity could span between 270-330 mOsm / kg. The buffer composition may comprise sodium salts, including but not limited to sodium chloride, sodium phosphate, and similar substances.

[0096] EXAMPLE 3

[0097] This example illustrates a dermal filler product in accordance with one embodiment as discussed herein. The composition is a sterile, slowly-absorbed, non-pyrogenic, clear, colorless gel implants. It is composed of Modified Agarose Polysaccharide (MAP) in phosphate buffered saline (PBS) and contains 3 mg / mL lidocaine hydrochloride (0.3 % w / v). Agarose is a polysaccharide extracted from red algae and MAPs are derivatives of agarose, that are chemically modified.

[0098] The composition is supplied in a prefilled plastic syringe and the content of syringe is steam sterilized. In some embodiments, one syringe can be packed in a pouch and the needles delivered in a box separately. In another embodiment, one syringe can be packaged in a blister, with two needles. The composition has pH of 6.8 to 7.2, osmolarity of 300-345 mOsm / kg and G’ in the range of 3400-5000 Pa and G” of 300-450 Pa.

[0099] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0100] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0101] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0102] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0103] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0104] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0105] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0106] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0107] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMSWhat is claimed is:

1. A composition, comprising: a modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa; an anesthetic; and a bioburden of no more than 100 CFU / ml.

2. The composition of claim 1, wherein the modified agarose polysaccharide is present in a gel.

3. The composition of claim 2, wherein the gel is a hydrogel.

4. The composition of any one of claims 1-3, wherein the modified agarose polysaccharide has a weight average molecular weight of between 21 kDa and 200 kDa.

5. The composition of any one of claims 1-4, wherein the modified agarose polysaccharide has a polydispersity index of between 1.5 and 3.2.

6. The composition of any one of claims 1-5, wherein the modified agarose polysaccharide has a structure:wherein at least 10% of the R moieties in the structure are entities selected from the group consisting of carboxylate, halogen, sulfate, sulfonate, and phosphorylate.

7. The composition of claim 6, wherein at least 90% of the R moieties in the structure are entities selected from the group consisting of hydroxide, carboxylate, halogen, sulfate, sulfonate, and phosphorylate.

8. The composition of any one of claims 6 or 7, wherein at least 10% of the R moieties are carboxylate moieties.

9. The composition of any one of claims 6-8, wherein at least 10% of the R moieties are halogen moieties.

10. The composition of any one of claims 6-9, wherein at least 10% of the R moieties are sulfate moieties.

11. The composition of any one of claims 6-10, wherein at least 10% of the R moieties are sulfonate moieties.

12. The composition of any one of claims 6-11, wherein at least 10% of the R moieties are phosphorylate moieties.

13. The composition of any one of claims 1-12, wherein at least 50% of the modified agarose polysaccharide exhibits an at least partial beta-sheet structure.

14. The composition of any one of claims 1-13, wherein the modified agarose polysaccharide is prepared by oxidization an alcohol on agarose.

15. The composition of any one of claims 1-14, wherein the modified agarose polysaccharide is heat-treated modified agarose polysaccharide.

16. The composition of any one of claims 1-15, wherein the modified agarose polysaccharide has been exposed to a temperature of at least 100 °C.

17. The composition of any one of claims 1-16, wherein the modified agarose polysaccharide has been autoclaved.

18. The composition of any one of claims 1-17, wherein the bioburden is no more than 100 CFU / ml.

19. The composition of any one of claims 1-18, wherein the composition further comprises a pH buffer.

20. The composition of any one of claims 1-19, wherein the pH buffer is phosphate-buffered saline.

21. The composition of any one of claims 1-20, wherein the pH buffer is substantially free of potassium.

22. The composition of any one of claims 1-21, wherein the composition has a potassium concentration of less than 10 millimolar.

23. The composition of any one of claims 1-22, wherein the anesthetic is selected from the group consisting of lignocaine (lidocaine), bupivacaine, butanilicaine, carticaine, cinchocaine (dibucaine), clibucaine, ethylparapiperidinoacetylaminobenzoate, etidocaine, mepivacaine, oxethazaine, prilocaine,ropivacaine, tolycaine, trimecaine, vadocaine, articaine, levobupivacaine, amylocaine, cocaine, propanocaine,clormecaine, cyclomethycaine, proxymetacaine, amethocaine (tetracaine), benzocaine, butacaine, butoxycaine, butyl aminobenzoate, chloroprocaine, dimethocaine (larocaine), oxybuprocaine, piperocaine, parethoxycaine, procaine (novocaine), propoxycaine, tricaine, or combinations thereof.

24. The composition of any one of claims 1-23, wherein the anesthetic comprises lidocaine.

25. The composition of claim 24, wherein no more than 10 mol% of the lidocaine is hydrolyzed.

26. The composition of any one of claims 1-25, wherein the composition further comprises hyaluronic acid.

27. The composition of any one of claims 1-26, wherein the composition further comprises a mineral.

28. The composition of any one of claims 1-27, wherein the composition further comprises hydroxyapatite.

29. The composition of any one of claims 1-28, wherein the composition further comprises a vitamin.

30. The composition of any one of claims 1-29, wherein the composition further comprises a peptide.

31. A syringe comprising a barrel, the barrel containing the composition of any one of claims 1-30.

32. The syringe of claim 31, further comprising a needle attached to the barrel.

33. The syringe of claim 32, wherein the needle has an inner diameter of 0.9 mm or less.

34. The syringe of any one of claims 31-33, further comprising a cannula attached to the barrel.

35. A composition, comprising: a modified agarose polysaccharide having a shear- modulus G’ of between 10 Pa and 20 kPa;an anesthetic; and an endotoxin concentration of less than 0.5 EU / ml.

36. A composition, comprising: a modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa; an anesthetic; and an endotoxin concentration of less than 20 EU / ml.

37. A device, comprising: a syringe comprising a barrel, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, wherein the composition exhibits a bioburden of no more than 100 CFU / ml.

38. The device of claim 37, further comprising a needle attached to the barrel.

39. The device of claim 38, wherein the needle has an inner diameter of 0.9 mm or less.

40. The device of any one of claims 38 or 39, wherein the needle has an inner diameter of 0.6 mm or less.

41. The device of any one of claims 37-40, further comprising a cannula attached to the barrel.

42. The device of any one of claims 37-41, wherein the barrel comprises glass.

43. The device of any one of claims 37-42, wherein the barrel comprises a polymer.

44. The device of claim 43, wherein the polymer comprises a cyclic olefin copolymer.

45. The device of any one of claims 37-44, wherein the composition further comprises an anesthetic.

46. The device of any one of claims 37-45, wherein the modified agarose polysaccharide is present in a gel.

47. The device of any one of claims 37-46, wherein the modified agarose polysaccharide has a average molecular weight of between 21 kDa and 180 kDa.

48. The device of any one of claims 37-47, wherein the modified agarose polysaccharide has a polydispersity index of between 1.5 and 3.2.

49. The device of any one of claims 37-48, wherein the modified agarose polysaccharide has a structure:wherein at least 10% of the R moieties in the structure are entities selected from the group consisting of carboxylate, halogen, sulfate, sulfonate, and phosphorylate.

50. The device of any one of claims 37-49, wherein at least 90% of the R moieties in the structure are entities selected from the group consisting of hydroxide, carboxylate, halogen, sulfate, sulfonate, and phosphorylate.

51. The device of any one of claims 37-50, wherein at least 50% of the modified agarose polysaccharide exhibits an at least partial beta-sheet structure.

52. The device of any one of claims 37-51, wherein the modified agarose polysaccharide is prepared by oxidization an alcohol on agarose.

53. The device of any one of claims 37-52, wherein the composition further comprises a pH buffer.

54. The device of any one of claims 37-53, wherein the composition has a potassium concentration of less than 10 millimolar.

55. The device of any one of claims 37-54, wherein the anesthetic comprises lidocaine.

56. The device of any one of claims 37-55, wherein the composition further comprises hyaluronic acid.

57. A device, comprising: a syringe comprising a barrel, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, wherein the modified agarose polysaccharide has an endotoxin concentration of less than 0.5 EU / ml.

58. A device, comprising: a syringe comprising a barrel, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, wherein the modified agarose polysaccharide has an endotoxin concentration of less than 20 EU / ml.

59. A method, comprising: injecting, into a subject, a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, wherein the composition exhibits a bioburden of no more 100 CFU / ml.

60. The method of claim 59, wherein the subject is diagnosed with exhibiting hair loss.61 . The method of any one of claims 59 or 60, comprising injecting the composition into the face of a subject.

62. The method of any one of claims 59-61, comprising injecting the composition into a wrinkle in the skin of the subject.

63. The method of any one of claims 59-62, comprising injecting the composition into a wound in the skin of the subject.

64. The method of any one of claims 59-63, comprising injecting the composition into a portion of the skin that is dry.

65. The method of any one of claims 59-64, comprising injecting the composition into a portion of the skin that is sun-damaged.

66. The method of any one of claims 59-65, wherein the subject is diagnosed with a hyperpigmentation disorder.

67. The method of any one of claims 59-66, comprising injecting the composition into a portion of the skin that exhibits psoriasis.

68. The method of any one of claims 59-67, comprising injecting the composition into a portion of the skin that exhibits eczema.

69. The method of any one of claims 59-68, comprising injecting the composition into a portion of the skin that exhibits asteototic eczema.

70. The method of any one of claims 59-69, comprising injecting the composition into the cutis of the skin.71 . The method of any one of claims 59-70, comprising injecting the composition into the subcutis of the skin.

72. The method of any one of claims 59-71, comprising injecting the composition into the body of the subject.

73. The method of any one of claims 59-72, comprising injecting the composition into the hand of the subject.

74. A method, comprising: injecting, into a subject, a comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 0.5 EU / ml.

75. A method, comprising: injecting, into a subject, a comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 20 EU / ml.

76. A kit, comprising: a syringe comprising a barrel, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, wherein the modified agarose polysaccharide exhibits a bioburden of no more 100 CFU / ml.

77. The kit of claim 76, further comprising a needle.

78. The kit of claim 77, wherein the needle has an inner diameter of 0.9 mm or less.

79. The kit of any one of claims 76-78, further comprising a cannula.

80. A kit, comprising: a syringe comprising a barrel, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 0.5 EU / ml.

81. A kit, comprising: a syringe comprising a barrel, the barrel containing a composition comprising modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 20 EU / ml.

82. A device, comprising: a syringe comprising a barrel, the barrel containing autoclaved modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa.

83. The device of claim 82, wherein the device further comprises a needle attached to the syringe.

84. The device of claim 83, wherein the needle has an inner diameter of 0.9 mm or less.

85. The device of any one of claims 82-84, wherein the device further comprises a cannula attached to the syringe.

86. A device, comprising: a syringe comprising a barrel and a needle, the barrel containing heat-treated modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa.

87. The device of claim 86, wherein the heat-treated modified agarose polysaccharide has been exposed to a temperature of at least 100 °C.

88. Use of a composition for the treatment of a subject, wherein the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, wherein the modified agarose polysaccharide exhibits a bioburden of no more than 100 CFU / ml.

89. The use of the composition of claim 88, for improving the appearance of skin of the subject.

90. The use of the composition of claim 88, for preventing hair loss in the subject.

91. The use of the composition of claim 88, for treating hair loss in the subject.

92. The use of the composition of claim 88, for filling wrinkles of the subject.

93. The use of the composition of claim 88, for contouring the skin of the subject.

94. The use of the composition of claim 88, for healing a wound of the subject.

95. The use of the composition of claim 88, for treating dry skin of the subject.

96. The use of the composition of claim 88, for treating sun-damaged skin of the subject.

97. The use of the composition of claim 88, for treating a hyperpigmentation disorder in the subject.

98. The use of the composition of claim 88, for treating, in the subject, a disease that has inflammation as a component of the disease process.

99. The use of the composition of claim 88, for treating psoriasis of the subject.

100. The use of the composition of claim 88, for treating eczema of the subject.

101. The use of the composition of claim 88, for treating astcototic eczema of the subject.

102. The use of the composition of claim 88, for adding volume to the subject.

103. The use of the composition of claim 88, for treating wrinkles in the subject.

104. The use of the composition of claim 88, for adding volume in a hand of the subject.

105. Use of a composition for the treatment of a subject, wherein the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 0.5 EU / ml.

106. Use of a composition for the treatment of a subject, wherein the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 20 EU / ml.

107. Use of a composition for the manufacture of a medicament for the treatment of a medical condition in a subject, wherein the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa, wherein the modified agarose polysaccharide exhibits a bioburden of no more than 100 CFU / ml.

108. Use of a composition for the manufacture of a medicament for the treatment of a medical condition in a subject, wherein the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 0.5 EU / ml.

109. Use of a composition for the manufacture of a medicament for the treatment of a medical condition in a subject, wherein the composition comprises modified agarose polysaccharide having a shear modulus G’ of between 10 Pa and 20 kPa and an endotoxin concentration of less than 20 EU / ml.

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