WO2021141595A1

WO2021141595A1 - Preparation and preservation of collagen microbubbles

Info

Publication number: WO2021141595A1
Application number: PCT/US2020/013055
Authority: WO
Inventors: Jameel FESHITAN
Original assignee: Advanced Microbubbles Laboratories Llc
Priority date: 2020-01-10
Filing date: 2020-01-10
Publication date: 2021-07-15

Abstract

Compositions, methods, and utilities related to stabilized collagen microbubbles. Collagen microbubbles may be produced and provided in a composition comprising a stabilizer to promote longevity of the collagen microbubbles in the composition. The stabilizer may comprise a fatty acid such as steric acid. Additional stabilizers may be provided. Furthermore, additives may be provided such as one or more healing promotion agents. In turn, a composition comprising collagen microbubbles may be facilitated that may have usefulness in a number of applications including, for example, wound care.

Description

PREPARATION AND PRESERVATION OF COLLAGEN MICROBUBBLES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional App. No. 62/791,423 entitled "PREPARATION AND PRESERVATION OF COLLAGEN MICROBUBBLES" filed on 11 January 2019, the entirety of which is incorporated by reference herein.

BACKGROUND

Collagen is an abundantly present protein found in the body of mammals. Collagen comprises a helical structure of amino acids that form elongated fibrils in the extracellular space and in various connective tissues of mammals. Collagen can comprise 25% to 35% of the total protein content of an animal's body. A number of types of collagen have been described including Type I (e.g., as found in the skin, tendons, vasculature, organs, and bones of mammals), Type II (e.g., as found in cartilage of mammals), Type III (e.g. comprising reticular fibers of mammals), Type IV (e.g. comprising basil lamina), and Type V (e.g. comprising cell surfaces, hair, and placentas of mammals). Type I is the most prevalent type of collagen in the human body, although at least 28 types of collagen have been identified and described.

Collagen has been found to have application in medical treatment. Accordingly, a number of medical treatment applications have been contemplated that utilize collagen for various purposes. Notable among the proposed uses of collagen in a medical treatment application is the use of collagen in wound care. Specifically, collagen in an activated or hydrolyzed form may be particularly useful in wound treatment. It has been found that collagen plays an important role in most, if not all, phases of regular wound healing. In turn, the introduction of supplemental collagen to a wound site may provide benefits in all phases of the wound healing process. For instance, collagen has been found to promote wound closure and may assist in maintaining sterility of the wound as collagen is naturally resistant to bacteria. Other medical applications that employ collagen for beneficial therapeutic purposes have been proposed in which supplemental collagen (i.e., collagen that is not naturally occurring within the body) may be introduced to the body in various forms.

In relation to wound care, it has also been found that oxygen may be beneficial to promote healing. Like collagen, it has been found that oxygen plays a critical role nearly all stages of wound healing. Flowever, means for efficient introduction of oxygen to a wound site have remained difficult to find. Proposed approaches that utilize oxygen to promote wound healing include hyperbaric oxygen therapy and topical oxygen therapy. Flowever, the efficiency, effectiveness, and convenience of such approaches are limited such that widespread adoption has not been achieved. In view of the foregoing, while it has generally been recognized that collagen and/or oxygen may be beneficial for certain medical treatment applications, the ability to efficiently, effectively, and conveniently take advantage of the noted benefits has been limited. As such, the need persists for improvements in the products and methods that may be used to leverage the recognized benefits.

SUMMARY

Accordingly, the present disclosure relates to compositions, methods, and other utilities that include collagen microbubbles that are stabilized for increased longevity of the collagen microbubbles. In turn, the present disclosure facilitates benefits in relation to use of collagen microbubbles in a number of medical treatment applications in which delivery of supplemental collagen creates advantages. One particular example is in the application of wound care.

It is presently recognized that the use of collagen microbubbles may provide advantages as a means for delivery of supplemental collagen and other beneficial material. However, it is also recognized that traditional approaches to providing collagen microbubbles suffered in that collagen microbubbles have extremely limited longevity. In this regard, it has traditionally been the case that collagen microbubbles may degrade prior to use such that an effective amount of microbubbles is difficult to realize. For instance, in traditional approaches in which collagen microbubbles have been utilized, the stability of the collagen microbubbles may result in significant degradation of the microbubbles within days or even hours after production. As such, the inability to preserve or stabilize collagen microbubbles to increase the longevity of the microbubbles in prior approaches represents a limitation on the use of collagen microbubbles in many applications as the microbubbles comprising a collagen shell tend to deteriorate prior to being used.

Accordingly, it is presently recognized that use of one or more stabilizers may be used to provide enhanced preservation of collagen microbubbles such that the use of collagen microbubbles may be expanded into new applications because of the enhanced stability of the collagen microbubbles. While a number of stabilizers are contemplated herein, it has been found that a fatty acid in a composition comprising a population of collagen microbubbles may significantly prolong the stability of collagen microbubbles over time. In particular, stearic acid has been found to significantly prolong the stability over time of collagen microbubbles in a cake of microbubbles present in a composition. While stearic acid may be of particular interest, other fatty acids are contemplated for use in stabilizing collagen microbubbles.

Additional stabilizers may also be provided that may enhance the stability of the collagen microbubbles in a composition. In certain embodiments, an additional stabilizer may be provided that results in synergistic benefits to the stability of collagen microbubbles. As will be shown herein, such additional stabilizers may have little stabilization effect in isolation but may provide significant improvements to stability of collagen microbubbles when used in conjunction with a fatty acid. As such, additional stabilizers beyond a fatty acid are contemplated that may be used in isolation or in combination with a fatty acid. Examples of such additional or alternative stabilizers include polyols, monosaccharides, polysaccharide, phospholipids, phosphoproteins, glycoproteins, glycolipids, combinations thereof, and/or components thereof. Specifically, stabilizing additives that may, individually or in combination, be provided may include, dextrose, medical grade honey, lecithin, pectin, xanthan gum, cholesterol, casein, gum Arabic, and/or fatty alcohols.

Further still, one or more healing promotion agents may be included with the collagen microbubbles in a composition. Such a healing promotion agent may be particularly advantageous when the collagen microbubble composition is to be used in the application of wound care. In one embodiment, the healing promotion agent may comprise oxygen, which may be entrained in a fluid core of the microbubbles. While it has been recognized that collagen and oxygen independently play important roles in wound care and may provide advantageous medical benefits, the use of collagen microbubbles in combination with oxygen may allow for the benefits of both collagen and oxygen to be provided to a wound to which the collagen microbubbles are applied. That is, the fluid core of the collagen microbubbles may comprise oxygen (e.g. essentially pure oxygen) that may be utilized to deliver oxygen to the wound site. Oxygen delivery to a wound site may be more efficient when it is supplied in the fluid core of a microbubble due to the much greater interfacial surface area to volume ratio and larger gas transfer coefficient of oxygen filled microbubbles as opposed to macroscopic oxygen such as that used in traditional therapies like hyperbaric oxygen therapy and topical oxygen therapy. Accordingly, oxygen provided in the fluid core of the collagen microbubbles may comprise a healing promotion agent that is concurrently delivered to a wound site with collagen when the collagen microbubbles are applied to a wound site. Oxygen filled collagen microbubbles may also provide advantages in other applications other than wound care (e.g., including treatment of acute respiratory distress syndrome or hypoxia) as will be discussed in greater detail below. Furthermore, use of collagen and/or oxygen in other applications may provide benefits including enhanced metabolic performance or as an analgesic. Other healing promotion agents in addition to or as an alternative to oxygen may also be provided with the collagen microbubbles as will be described in greater detail below.

In view of the foregoing, the present disclosure relates to compositions and related methodology for microbubbles that may comprise collagen in the microbubble structure.

Specifically, microbubbles may be produced such that the microbubbles comprise collagen shells that surround a fluid core of the microbubble. It is proposed that the use collagen microbubbles may provide advantages in relation to delivery mechanisms for introduction of collagen and/or other substances (e.g., oxygen) to the body of a mammal. For example, use of collagen microbubbles may allow for more effective wound treatment to promote healing of a wound in the presence of collagen microbubbles. Furthermore, other approaches are contemplated that utilize collagen microbubbles to provide various advantageous effects as will be discussed herein. Moreover, a stabilizer and/or wound healing promotion agent may be provided. The stabilizers and/or healing promotion agents may be provided in the composition comprising the collagen microbubbles, may comprise the shell structure of the collagen microbubbles, and/or may be contained in the fluid core of the collagen microbubbles. Additional components of a composition comprising collagen microbubbles may be provided in an aqueous solution from which the microbubbles are formed such that the other components are in the composition from the time the collagen microbubbles are formed or the additional components may be added to a composition of collagen microbubbles after the microbubbles are formed.

Furthermore, while wound care is one contemplated application of stabilized collagen microbubbles, other applications are also currently contemplated. These may be related to wound care such as, for example, use of collagen microbubbles in tissue engineering (e.g., in which collagen microbubbles may be used as a collagen scaffold for cell growth), use of collagen microbubbles to treat hypoxia, and/or use of collagen microbubbles as an analgesic. Other applications such as improved metabolic performance may be achieved by ingestion of collagen microbubbles. In this regard, stabilized collagen microbubbles may be provided the form of a supplement to enhance performance. Specifically, stabilized collagen microbubbles may be provided in a sports beverage.

Accordingly, a first aspect of the present disclosure includes a stabilized collagen microbubble composition. The composition includes an aqueous media and a population of collagen microbubbles in the aqueous media. The collagen microbubbles each include a collagen shell and a gas core. The composition further includes a fatty acid in the aqueous media. Specifically, the fatty acid acts to preserve the longevity of the collagen microbubbles in the aqueous media.

A number of feature refinements and additional features are applicable to the first aspect. These feature refinements and additional features may be used individually or in any combination.

As such, each of the following features that will be discussed may be, but are not required to be, used with any other feature or combination of features of the first aspect.

For instance, in an embodiment, the fatty acid may be stearic acid. Flowever, in alternative embodiments, the fatty acid may be additionally or alternatively selected from the group consisting of palmitic acid, oleic acid, and lauric acid. The collagen shell may be at least one of type 1 collagen, type 3 collagen, activated collagen, hydrolyzed collagen, or combinations thereof. Furthermore, the collagen microbubbles may comprise not less than about 2.5 weight percent of the composition and not greater than about 40 weight percent of the composition. The fatty acid may comprise not less than about 0.5 weight percent of the composition and not greater than about 30 weight percent of the composition. In other embodiments, the fatty acid may comprise not less than about 1 weight percent of the composition, not less than 5 weight percent of the composition, not less than 10 weight percent of the composition, or not less than 25 weight percent of the composition. In a particular embodiment, the fatty acid may comprise not less than about 1 weight percent and not greater than about 10 weight percent of the composition.

In an embodiment, the composition may further include another stabilizer. The stabilizer may comprise a polyol, monosaccharide, polysaccharide, phospholipid, phosphoprotein, glycoprotein, glycolipid, components thereof, and/or combinations thereof. The additional stabilizer may comprise a viscosity enhancer such as glycerol, dextrose, and/or medical grade honey. These components may be provided in liquid form and may comprise not less than about 1 volume percent of the aqueous media and not greater than about 40 volume percent of the aqueous media. In other embodiment, the additional stabilizer may comprise lecithin, pectin, xanthan gum, cholesterol, casein, gum Arabic, and/or fatty alcohol. The additional stabilizer may be provided in powdered form. In any regard, the additional stabilizer may comprise not less than about 1 weight percent of the composition and not greater than about 40 weight percent of the composition . In one particular embodiment, the composition may comprise stearic acid and lecithin in combination, which has been found to significantly improve the stability of the collagen microbubbles in such a composition. In another embodiment, the composition may include stearic acid and pectin, which has also been shown to provide synergistic effects to improve the stability of collagen microbubbles in such a composition.

In an embodiment, the gas core of the collagen microbubbles may consist of oxygen. That is, pure or essentially pure (e.g., 90% pure, 95% pure, or even 99% pure) oxygen may be provided within the collagen microbubble. In this regard, the oxygen contained by the collagen microbubbles may comprise a healing promotion agent. However, in other embodiments, additional or alternative healing promotion agents may be provided. For example, in one embodiment, the healing promotion agent may be L-proline. In any regard, the healing promotion agent may comprise not less than about 1 weight percent of the composition and not greater than about 6 weight percent of the composition. In an embodiment, the population of the collagen microbubbles may include a concentrated population generated by centrifugal washing of a disperse microbubble solution. In this regard, the population of the collagen microbubbles may be a polydisperse population of collagen microbubbles comprising a majority by volume of collagen microbubbles in a size of not less than about 0.5 pm and not greater than about 30 pm. Alternatively, the collagen microbubbles may be isolated by size to provide a targeted size of microbubble in the composition.

A second aspect included herein is a method for producing a stabilized collagen microbubble composition. The method includes preparing a solution comprising at least activated collagen and a fatty acid in an aqueous media. Also, the method includes introducing a gas phase at a surface of the solution at a gas phase pressure greater than atmospheric pressure. In turn, the method includes producing a population of collagen microbubbles by exciting the solution. Specifically, the population of collagen microbubbles are provided in the aqueous media comprising the fatty acid for preservation of the longevity of the collagen microbubbles in the aqueous media.

A number of feature refinements and additional features are applicable to the second aspect. These feature refinements and additional features may be used individually or in any combination. As such, each of the following features that will be discussed may be, but are not required to be, used with any other feature or combination of features of the second aspect.

For example, in an embodiment, the method may further include heating the solution under mixing to clarify the solution prior to microbubble formation. As such, the method may include cooling the solution to a production temperature above a gel-forming temperature of the solution after clarifying the solution. That is, the production temperature may be above the gel-forming temperature but below the temperature to which the solution is heated to achieve good solubility. These steps may promote microbubble formation by initially providing good solubility of the collagen in the aqueous media to promote mixing, while enhancing bubble formation at a temperature nearer the gel formation temperature (e.g., where the solution is more viscous to promote bubble formation). In this regard, the aqueous solution may be is heated to a temperature at which the collagen becomes soluble in the solution. Thereafter the method may include cooling the population of collagen microbubbles prior to producing the microbubbles to a temperature above the gel-forming temperature. Further still, the method may include cooling the population of collagen microbubbles after the producing to a temperature at or below the gel-forming temperature. As may be appreciated, this may assist in microbubble stability as the composition may be at or below the gel-forming temperature at which the collagen forming the microbubbles becomes more stable as it gels in the composition. In this regard, the method may include storing the solution at a temperature at or below the gel-forming temperature. In an embodiment, the gel-formation temperature may be between about 2 degrees Celsius and 15 degrees Celsius. In further embodiments, additives may be provided to modify the gel- formation temperature of the composition. Specifically, a gel-temperature modifier may be introduced to raise the gel-temperature to at or above room temperature (roughly 20-25 degrees Celsius). In this regard, the composition comprising the collagen microbubbles may be below the gel- formation temperature when stored at room temperature. This may provide convenience for storage of the composition without requiring refrigeration.

The excitation of the solution may be performed in a number of different manners. For instance, the exciting the solution may include sonicating the gas-liquid interface between the solution and the gas phase. However, other approaches to excitation of the solution to produce the microbubbles may be provided, which may include for example, mechanical agitation. The mechanical agitation may be applied to the aqueous solution and the containment vessel of the aqueous solution. Further still, excitation may include action on the fluid alone to produce microbubbles. This may include blending the aqueous solution using a blender, colloid mill, homogenizer, or other device to excite the fluid and produce the microbubbles.

In an embodiment, the gas phase consists of oxygen. That is, pure or essentially pure oxygen may be provided within the collagen microbubble. In this regard, the oxygen contained by the collagen microbubbles may comprise a healing promotion agent. Further still, the solution may further comprise a healing promotion agent comprising L-proline.

Furthermore, the solution may also include an additional stabilizer that may be present in the solution prior to production of the microbubbles or introduced after production or may be added after microbubble production. For instance, the solution may include any type of additional stabilizer as addressed above in relation to the first aspect. Specifically, the solution may include a stabilizer selected from the group consisting of glycerol, dextrose, medical grade honey, lecithin, pectin, xanthan gum, cholesterol, casein, gum Arabic, and fatty alcohol.

In an embodiment, the method may also include centrifuging the population of the collagen microbubbles to produce a concentrated supernatant cake of collagen microbubbles in the aqueous solution. Additionally or alternatively, the method may include isolating a targeted size of microbubble (e.g., using centrifugation).

As will be described in detail herein, the method of the second aspect may also include various applications of the composition comprising the collagen microbubbles. For instance, the method may also include applying the population of the collagen microbubbles to a wound of a patient in an effective amount to induce healing of the wound. Additionally, the method may include adding the population of collagen microbubbles to a beverage. The method may include using the population of collagen microbubbles in tissue engineering (e.g., including use of the population of collagen microbubbles as a collagen scaffold for cell generation). As another option, the method may include using the population of collagen microbubbles to treat hypoxia. Further still, the method may include using the population of collagen microbubbles as a pain remedy.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts a progressively more detailed description of collagen.

Figure 2 depicts an embodiment of a system for generating a composition comprising a population of collagen microbubbles.

Figure 3 depicts a graph representing a size distribution of an embodiment of the composition comprising a population of collagen microbubbles.

Figure 4 depicts a before and after separation, that may be utilized to generate a cake of collagen microbubbles.

Figure 5 depicts an embodiment of the separation column with targeted size isolation microbubbles in the composition.

DETAILED DESCRIPTION

The following description is not intended to limit the invention to the forms disclosed herein. Consequently, variations and modifications commensurate with the following teachings, skill and knowledge of the relevant art, are within the scope of the present invention. The embodiments described herein are further intended to explain modes known of practicing the invention and to enable others skilled in the art to utilize the invention in such, or other embodiments and with various modifications required by the particular applications(s) or use(s) of the present invention.

As described above, is presently recognized that a composition comprising a population of collagen microbubbles may be used in a number of applications to provide therapeutic, performance, or other benefits. With reference to Fig. 1, a depiction of a collagen structure 10 is represented in a number of different scales. Specifically, the collagen structure 10 depicted in Figure 1 includes a bundle of collagen fibers 12. Each collagen fiber 12 includes collagen fibrils 14. The collagen fibrils 14 are comprised of collagen molecules also referred to as tropocollagen. Each collagen molecule or tropocollagen 16 comprises a triple helix of procollagen chains 18. The procollagen chains 18 comprise amino acids wound together to form a triple helix of the collagen molecule 16. The details of the procollagen chain 18 are shown in Fig. 1 and may generally include any one of a number of amino acid sequences. Common examples of such amino acid sequences include the depicted sequence of hydroxyproline 20, glycine 22, and proline 24. Other sequences of collagen may include glycine-proline-X or glycine-X-hydroxyproline where X is any amino acid other than glycine, proline, or hydroxyproline. In this regard, the specific sequence of amino acids may be one factor that determines type of collagen that may be provided. Examples of amino acids that may be included in an amino acid sequence of collagen include glycine, proline, alanine, hydroxyproline, glutamic acid, arginine, aspartic acid, serine, lysine, leucine, valine, threonine, phenylalanine, isoleucine, hydroxylian, methionine, histidine, tyrosine, cysteine, and/or tryptophan.

Accordingly, any number of different types of collagen may be provided. For example, there have been at least 28 different types of collagen identified, although the most common include Type I, Type II, Type III, Type IV, and Type V. Collagen may also be hydrolyzed, which may be referred to as gelatin when provided in the hydrolyzed form. In this regard, the hydrolyzed, low molecular weight form of different types of collagen may be provided. Any appropriate type of collagen may be utilized in the process and approaches described herein, although in a particular embodiment, it is envisioned that collagen Type I, Type II, Type III, Type IV, and Type V may be utilized.

With further reference to Fig. 2, a system 100 is depicted that may be utilized to prepare a composition 200 of collagen microbubbles. The system 100 may include an aqueous solution 102. The aqueous solution 102 may comprise an aqueous media in which various constituents may be provided in solution to form the aqueous solution 102. For example, collagen may be provided in the aqueous media 102 in preparation for production of a population of collagen microbubbles. A fluid 106 may be provided in a headspace 108 of a container 110 in which the aqueous solution 102 comprising the collagen 104 is disposed. For example, the fluid 106 may comprise a gas phase that is introduced by way of a gas source 112. Any number of different gases 106 may be utilized in the production of the collagen microbubbles. Specific examples of gas 106 may be utilized include atmospheric air, oxygen (e.g., pure or substantially pure oxygen), perfluorobutane (PFB), or other appropriate gas. The gas 106 may be provided in headspace 108 at a pressure elevated from that of atmospheric pressure. Specifically, the gas 106 may be provided at an elevated pressure such as 2 bar, 5 bar, 10 bar, 15 bar, or even 20 bar or greater. Accordingly, a liquid-gas interface may be defined between the aqueous solution 102 and headspace 108 comprising a high-pressure gas 106. Accordingly, and as will be described in greater detail below, the excitement of the aqueous solution 102 may cause production of microbubbles at the liquid-gas interface to form microbubbles comprising a shell from the collagen in the aqueous solution 102 with a gas core comprising the gas 106.

The collagen may be provided in the aqueous solution 102 in an amount comprising not less than 1.0 weight percent, not less than 2.5 weight percent, not less than 3.0 weight percent, not less than 5.0 weight percent, or even not less than 10.0 weight percent of the aqueous solution. In addition, the collagen may be provided in an amount comprising not greater than 60 weight percent, not greater than 50 weight percent, not greater than 40 weight percent, or even not greater than 30 weight percent. In this regard, collagen may be provided in the aqueous solution 102 in a range of not less than 1.0 weight percent and not greater than 60 weight percent of the aqueous solution 102, any range of not less than 2.5 weight percent and not greater than 40 weight percent of the aqueous solution 102, or not less than 5.0 weight percent and not greater than 30 weight percent of the aqueous solution 102.

As described above, the collagen may be of any type. In a preferred embodiment, collagen may comprise Type I or Type III collagen. Furthermore, the collagen may be provided in hydrolyzed or activated form.

The aqueous solution 102 may also include at least one stabilizer that may help to preserve the longevity of collagen microbubbles that are formed from the aqueous solution 102. The stabilizer may be present in the aqueous solution 102 prior to formation of the collagen microbubbles from the aqueous solution 102 or may be added to the composition 200 once the collagen microbubbles have been formed.

In an embodiment, the stabilizer may comprise a fatty acid. One example of such a fatty acid may include steric acid. Additionally or alternatively, the fatty acid may include palmitic acid, oleic acid, and/or lauric acid, although other fatty acids, whether saturated or unsaturated, may be provided. Furthermore, the stabilizing agent may comprise constituent components of fatty acids such as triglycerides, phospholipids, and/or cholesterol esters. In any regard, the stabilizer comprising a fatty acid may be provided the aqueous solution 102 in an amount of about 1 weight percent of the aqueous solution 102. In other embodiments, the fatty acid may be provided in the aqueous solution 102 an amount of not less than about 0.5 weight percent and not greater than about 30 weight percent or not less than about 1.0 weight percent and not greater than about 10 weight percent. The fatty acid provided the aqueous solution 102 may include essentially a single fatty acid such as steric acid or may comprise combinations of various fatty acids without limitation.

The aqueous solution 102 may further include an additional stabilizer in addition to or as an alternative to the fatty acid. Examples of other stabilizers that may be provided include polyols, monosaccharides, polysaccharide, phospholipids, phosphoproteins, glycoproteins, glycolipids, and/or components thereof. The additional stabilizer may comprise a viscosity enhancer such as glycerol, dextrose, and/or medical grade honey. These components may be provided in liquid form and may comprise not less than about 1 volume percent of the aqueous media and not greater than about 40 volume percent of the aqueous media. Other additional stabilizers that may, individually or in combination, be provided in the aqueous solution 102 may include, , lecithin, pectin, xanthan gum, cholesterol, casein, gum Arabic, and/or fatty alcohols. These additional stabilizers may be provided in powder form. In any regard, the additional stabilizers may be added in an amount of not less than 1.0 weight percent of the aqueous solution 102, not less than 1.5 weight percent of the aqueous solution 102, not less than 2.0 weight percent of the aqueous solution 102, not less than 2.5 weight percent of the aqueous solution 102, or even not less than 3.0 weight percent of the aqueous solution 102. Additionally or alternatively, the additional stabilizer may be added an amount not greater than 30 weight percent of the aqueous solution 102, not greater than 20 weight percent of the aqueous solution 102, not greater than 10 weight percent of the aqueous solution 102, or even not greater than 5.0 weight percent of the aqueous solution 102.

In one particular embodiment, the aqueous solution 102 and/or the resulting composition 200 containing the collagen microbubbles may comprise stearic acid and lecithin, which has been found to provide significant stabilization of the collagen microbubbles as will be shown in the examples below. Further still, a composition 200 may include stearic acid and pectin, which have also been found to provide significant improvements to the longevity of collagen microbubbles.

Furthermore, the aqueous solution 102 and/or the resulting composition 200 may include a healing promotion agent. The healing promotion agent may be introduced into the aqueous solution 102 prior to collagen microbubble production or to the composition 200 subsequent to collagen microbubble production. The healing promotion agent may, in an embodiment, comprise L-proline. Additionally or alternatively, the healing promotion agent may comprise oxygen, which may comprise the gas 106 introduced in the headspace 108 of the container 110, which is ultimately entrained in the collagen microbubbles once formed. In this regard, the oxygen comprising a gas core of the collagen microbubbles may comprise the healing promotion agent, which may be delivered via the collagen microbubbles in the aqueous solution 102. In any regard, the healing promotion agent may be provided in an amount not less than about 1.0 weight percent and not greater than about 60%.

With continued reference to Fig. 2, the system 100 may also include an actuator 120 that acts on the aqueous solution 102. The actuator 120 may excite the aqueous solution 102 so as to form a population of collagen microbubbles in the aqueous solution 102. The excitation of the aqueous solution 102 may include mechanical agitation of the system including the container 110 or may act solely on the aqueous fluid 102. Examples of actuators and processes that may be used to excite the aqueous solution 102 for the production of collagen microbubbles include, for example, a sonicator for sonification at the liquid gas interface, a mechanical agitator for shaking the aqueous solution 102, a dental amalgamator for acting on the aqueous solution 102, or an in-line homogenizer/colloid mill for acting on the aqueous solution 102. Prior to excitation, the aqueous solution 102 may be heated to promote solubility of the collagen in the aqueous media. In this regard, the aqueous solution 102 may be heated until solubility of the collagen is achieved and the solution clarifies. This may include heating the solution to a temperature above the gel-formation temperature of the collagen at which the collagen forms a slurry or semi-solid viscous gel. After elevating the temperature of the aqueous solution 102 to achieve solubility of the collagen, the microbubbles may be formed by excitation of the liquid-gas interface of the aqueous solution 102. Alternatively, the aqueous solution 102 may be cooled from the temperature used to achieve solubility to a reduced temperature that is still above the gel- formation temperature prior to excitation to produce the microbubbles. In any regard, once the microbubbles are formed, the solution may be cooled to below the gel-formation temperature for long term storage of the composition 200 comprising the collagen microbubbles. For instance, the gel-formation temperature may be between about 2 degrees Celsius and 15 degrees Celsius. Storage of the composition 200 at a temperature below the gel-formation temperature may assist in preserving the population of collagen microbubbles. In one embodiment, the composition 200 may be cooled to a temperature well below the gel-formation temperature, e.g., to a temperature below 4 degrees Celsius. In other embodiments, the composition 200 may be super cooled to a temperature below -10 degrees Celsius, below -20 degrees Celsius, below -50 degrees Celsius, or below -70 degrees Celsius..

In another embodiment, a gel-temperature modifier may be added to the composition 200 to adjust the gel-temperature of the composition 200. Specifically, it may be appreciated that storage of the composition 200 without requiring refrigeration may be desirable. As such, it may be advantageous to increase the gel-formation temperature to at or above room temperature (e.g., 20- 25 degrees Celsius). In this regard, when the composition 200 is at room temperature, the composition 200 may still be below the gel-formation temperature so as to further improve stability of the collagen microbubbles.

In this regard, after production of the collagen microbubbles, a composition 200 may be provided that includes the collagen microbubbles in the aqueous media that also includes one or more stabilizers in solution. It is been found that the utilization of a stabilizer as recited herein may significantly prolong the longevity of the collagen microbubbles in the composition 200, which are susceptible to rapid degradation in the absence of such stabilizers. Specific examples to demonstrate the increase in longevity are described below in greater detail.

Accordingly, with increased longevity of the collagen microbubbles, the capability for utilization of collagen microbubbles in various different applications may be facilitated. One such application may include the utilization of collagen microbubbles for wound treatment. In this regard, the composition comprising the population of collagen microbubbles may be applied to a wound of the patient in an effective amount to assist in the healing of the wound. Specifically, it is been found that introduction of collagen into the wound site may promote healing. Furthermore, as described above, introduction of oxygen to a wound site has also been determined to assist in wound healing. Accordingly, and in contrast to the traditional approaches for providing oxygen to a wound, delivery of oxygen by way of collagen microbubbles (e.g., which may release the oxygen locally at the wound site upon popping) may provide specific advantages. Furthermore, the utilization of healing promotion agent may further assist in relation wound care.

Other contemplated benefits of the composition comprising stabilized collagen microbubbles may also be realized. For example, the composition comprising population collagen microbubbles may be utilized in relation to tissue engineering. For instance, the collagen microbubbles may be utilized for a substrate or collagen scaffold on or about which cells may be regenerated or grown. That is, the collagen microbubbles may provide a structure that physically supports and fosters cell growth to provide a scaffold on which tissue may be grown. Yet another potential application may include utilization of the stabilized collagen microbubbles to treat hypoxia and/or acute respiratory distress syndrome. Specifically, the collagen microbubbles comprising oxygen as a gas core may be inhaled by a patient suffering from hypoxia and/or acute respiratory distress syndrome to provide delivery of supplemental oxygen with further beneficial effects from and/or healing promotion agent. Further still, it is contemplated that the collagen microbubbles may be utilized in analgesic either in connection with wound care or in isolation. Further still, it is contemplated that collagen microbubbles (e.g., potentially with oxygen gas cores) may provide performance benefits when ingested. In this regard, the collagen microbubbles may also be ingested by patient. For instance, the collagen microbubbles that have been stabilized as described herein provided in a sports beverage or the like.

With further reference to Fig. 3, a size distribution graph 300 is provided that describes the distribution of microbubble sizes in an exemplary composition comprising collagen microbubbles. As can be appreciated, the population of collagen microbubbles may be provided in a size of between about 0.5 pm and about 30 pm. For instance, the population of collagen microbubbles in the composition may comprise a polydisperse population of collagen microbubbles. Flowever, the collagen microbubbles may comprise a majority by volume of collagen microbubbles size of not less than about 0.5 micrometers and not greater than about 30 micrometers. This can be seen in the graph 300. Specifically, a large number of collagen microbubbles having a particle diameter of less than two micrometers may be provided. Flowever, as may be appreciated, the volume of such small microbubbles may contribute less to the total volume percentage thereof than larger particle diameters microbubbles such as those of diameter size of 4.0 micrometers and greater.

In an embodiment, the collagen microbubbles in the composition may be exposed to centrifugation to generate a supernatant cake 402 as shown in Fig. 4 comprising a concentration of microbubbles from the solution 200. Specifically, a separation column 400 may be provided that includes the composition 200 including a polydisperse population of collagen microbubbles. Upon exposure to centrifugation, a supernatant cake 402 comprising the microbubbles may be formed. An infranatant 404 may include residual collagen not forming microbubbles as well as the stabilizer and/or healing promotion agent. This infranatant 404 may be recycled for further use in an aqueous solution to generate additional microbubbles. Alternatively, the supernatant cake 402 comprising the collagen microbubbles may be transferred from the separation column 400 into a further aqueous media comprising the stabilizer and/or healing promotion agent. With further reference to Fig. 5, the collagen microbubble population 200 may be provided in a separation column 500 and exposed to centrifugal washing. This may isolate a target size of microbubble from the population into a supernatant cake 502 as described in US application publication number 2011/0300078, the entirety of which is incorporated by reference herein. An infranatant 504 may remain in the separation column 500 including microbubbles other than those of the targeted size.

Examples

Method preparing stabilized Collagen Shelled Microbubbles

1. The collagen solution is prepared by adding components comprising of 2.5 to 40 weight percent type I or III hydrolyzed/activated collagen, 1 weight percent stearic acid, 2 weight percent soy lecithin, 33 volume percent glycerol in 30 mL of aqueous media. Up to 5 weight percent of L-proline can be optionally incorporated in the mixture.

Notes: Glycerol serves as a humectant and reduces gas dissolution kinetics. Collagen serves as a component of the gas bubble shell and has wound healing benefits. Stearic acid is found to stabilize the collagen bubbles for longer storage and use. Soy lecithin may further enhance the stability of the collagen microbubbles e.g., when combined with stearic acid. L- proline is published to have wound healing benefits up to 5 weight percent.

Other additives to be considered included wound healing honey may include manuka honey.

2. The solution is heated under mixing until all components dissolve and the solution is clear.

3. The solution is cooled down to above its gel-forming temperature, e.g., by immersing in ice bath. Note: Gel-forming temperature is defined as the temperature when the collagen solution forms a semi-solid viscous gel or slurry. 4. The gas phase is introduced by flowing the gas phase over the surface of the solution (at high pressures), which drives out ambient air and cause an indentation of >½ inch at the surface.

Notes: The preferred gas core is oxygen because of its benefits for wound healing and treating hypoxia and hypoxemia. It is expected that the combined effect of collagen and oxygen would enhance the wound healing therapeutic benefits of this construct.

5. The collagen stabilized gas microbubbles are prepared by sonicating the gas liquid interface for at least 10 seconds.

6. The collagen microbubbles are concentrated by centrifugal washing in a syringe.

7. The concentrated collagen microbubbles are cooled to below the gel temperature and stored in a reduced temperature environment for later use.

Alternatively, the collagen microbubbles can be prepared via mechanical agitation (the shaking method) using a dental amalgamator or in-line homogenizer/colloid mill. The shaking method will potentially allow for easier GMP manufacturing.

As can be appreciated in the foregoing table, use of stearic acid in the composition significantly improves microbubble stability relative to a composition not including stearic acid. Furthermore, while lecithin provided in the composition with collagen microbubbles does not prolong stability, the combination of steric acid and lecithin provides significant improvement to stability of collagen microbubbles in the composition. Specifically, substantial preservation of the entire population of collagen microbubbles is maintained through seven day in the presence of 2 weight percent lecithin and 1 weight percent stearic acid. Furthermore, 60% of the microbubble population is maintained through four weeks with this combination. As such, it is evident that significant synergistic preservation is achieved through a combination of lecithin and stearic acid that is greater than the stability imparted by the individual use of such components alone. Furthermore, use of a combination of stearic acid and pectin also has shown beneficial synergistic improvements to collagen bubble stability. This is demonstrated in the results of Table 1 as well. For instance, a compositions including stearic acid and pectin maintained full bubble preservation through four weeks when refrigerated. Furthermore, use of stearic acid, pectin, and lecithin allowed full bubble stability at room temperature for a duration of at least 2 weeks, at which time the observation of this sample was discontinued. It is believed that this product would provide stability beyond the limited time of observation (e.g., up to at least 4 weeks per the other refrigerated samples).

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description is to be considered as exemplary and not restrictive in character. For example, certain embodiments described hereinabove may be combinable with other described embodiments and/or arranged in other ways (e.g., process elements may be performed in other sequences). Accordingly, it should be understood that only the preferred embodiment and variants thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.

Claims

What is claimed is:

1. A stabilized collagen microbubble composition, comprising: an aqueous media; a population of collagen microbubbles in the aqueous media , the collagen microbubbles each comprising a collagen shell and a gas core; and a fatty acid in the aqueous media, wherein the fatty acid acts to preserve the longevity of the collagen microbubbles in the aqueous media.

2. The composition of claim 1, wherein the fatty acid comprises stearic acid.

3. The composition of claim 1, wherein the fatty acid is selected from the group consisting of palmitic acid, oleic acid, and lauric acid.

4. The composition of any one of claims 1-3, wherein the collagen shell comprises at least one of type 1 collagen, type 3 collagen, activated collagen, hydrolyzed collagen, or combinations thereof.

5. The composition of any one of claims 1-4, wherein the collagen microbubbles comprise not less than about 2.5 weight percent of the composition and not greater than about 40 weight percent of the composition.

6. The composition of any one of claims 1-5, wherein the fatty acid comprises not less than about 0.5 weight percent of the composition and not greater than about 1.5 weight percent of the composition.

7. The composition of any one of claims 1-6, further comprising: a stabilizer selected from the group consisting of glycerol, dextrose, medical grade honey, lecithin, pectin, xanthan gum, cholesterol, casein, gum Arabic, and fatty alcohol.

8. The composition of claim 7, wherein the stabilizer comprises not less than about 1 volume percent of the aqueous media and not greater than about 40 volume percent of the aqueous media.

9. The composition of any one of claims 1-8, wherein the fatty acid comprises stearic acid and the composition further comprises lecithin.

10. The composition of any one of claims 1-8, wherein the fatty acid comprises stearic acid and the composition further comprises pectin.

11. The composition of claim 10, wherein the stearic acid is at a concentration of not less than 0.5 weight percent, the pectin is at a concentration of not less than 2.0 weight percent, and the lecithin is at a concentration of not less than 1 weight percent.

12. The composition of any one of claims 1-11, wherein the gas core consists of oxygen.

13. The composition of claim 12, further comprising: a healing promotion agent comprising L-proline.

14. The composition of claim 13, wherein the healing promotion agent comprises not less than about 1 weight percent of the composition and not greater than about 6 weight percent of the composition.

15. The composition of any one of claims 1-14, wherein the population of the collagen microbubbles comprise a concentrated population by centrifugal washing of a disperse microbubble solution.

16. The composition of any one of claims 1-15, wherein the population of the collagen microbubbles comprise a polydisperse population of collagen microbubbles comprising a majority by volume of collagen microbubbles in a size of not less than about 0.5m m and not greater than about 30 pm.

17. The composition of claim 1, wherein the fatty acid comprises steric acid at a concentration of not less than 0.5 weight percent of the composition, and the composition further comprises: pectin at a concentration of not less than about 2 weight percent; and lecithin at a concentration of not less than about 1 weight percent.

18. A pharmaceutical product comprising a stable collagen microbubble composition, the product comprising: a stabilized collagen microbubble composition according to any one of claims 1-17; and a sealed container having an internal container volume in which the stabilized collagen microbubble composition is disposed.

19. The pharmaceutical product of claim 18, wherein the stabilized collagen microbubble composition is in an injectable form and the sealed container comprises an injection device.

20. The pharmaceutical product of claim 19, wherein the injection device comprises a syringe.

21. The pharmaceutical product of claim 18, wherein the sealed container comprises a vial.

22. The pharmaceutical product of any one of claims 18-21, wherein the population of collagen microbubbles maintains not less than 90% of an initial volume of the population of collagen microbubbles when maintained in the sealed container at a temperature not greater than 4 °C for a duration not less than 4 weeks.

23. The pharmaceutical product of any one of claims 18-21, wherein the population of collagen microbubbles maintains not less than 90% of an initial volume of the population of collagen microbubbles when maintained in the sealed container at a temperature not greater than 25 °C for a duration not less than 4 weeks.

24. The pharmaceutical product of any one of claims 18-23, wherein the composition volume is substantially equal to the internal container volume.

25. A method for producing a stabilized collagen microbubble composition, comprising: preparing a solution comprising at least activated collagen and a fatty acid in an aqueous media; introducing a gas phase at a surface of the solution at a gas phase pressure greater than atmospheric pressure; and producing a population of collagen microbubbles by exciting the solution, wherein the population of collagen microbubbles are provided in the aqueous media comprising the fatty acid for preservation of the longevity of the collagen microbubbles in the aqueous media.

26. The method of claim 25, wherein the method further comprises: heating the solution under mixing to clarify the solution; and after clarifying the solution, cooling the solution to a production temperature above a gel forming temperature of the solution.

27. The method of claim 25, wherein the method further comprises: heating the solution under mixing to a temperature above a gel-forming temperature of the solution prior to the producing the population of collagen microbubbles.

28. The method of claim 27, wherein the solution is heated to a temperature at which the collagen becomes soluble in the solution.

29. The method of claim 28, further comprising: cooling the population of collagen microbubbles prior to the producing to a temperature above the gel-forming temperature.

30. The method of claim 29, further comprising: cooling the population of collagen microbubbles after the producing to a temperature below the gel-forming temperature.

31. The method of claim 30, further comprising: storing the solution at a temperature below the gel-forming temperature.

32. The method of claim 31, further comprising: maintaining not less than 90% of an initial volume of the population of collagen microbubbles when maintained in the sealed container at a temperature not greater than 4 °C for a duration not less than 4 weeks.

33. The method of claim 31, further comprising: maintaining not less than 90% of an initial volume of the population of collagen microbubbles when maintained in the sealed container at a temperature not greater than 25 oC for a duration not less than 4 weeks.

34. The method of any one of claims 25-33, wherein the exciting the solution comprises sonicating the gas-liquid interface between the solution and the gas phase.

35. The method of any one of claims 25-34, wherein the gas phase consists of oxygen.

36. The method of any one of claims 25-35, wherein the solution further comprises a stabilizer selected from the group consisting of glycerol, dextrose, medical grade honey, lecithin, pectin, xanthan gum, cholesterol, casein, gum Arabic, and fatty alcohol.

37. The method of any one of claims 25-35, wherein the fatty acid comprises stearic acid and the stabilizer comprises lecithin.

38. The method of any one of claims 25-35, wherein the fatty acid comprises stearic acid and the stabilizer comprises pectin and lecithin.

39. The method of any one of claims 25-38, wherein the solution further comprises a healing promotion agent comprising L-proline.

40. The method of any one of claims 25-39, further comprising: centrifuging the population of the collagen microbubbles to produce a concentrated supernatant cake of collagen microbubbles in the aqueous solution.

41. The method of any one of claims 25-40, further comprising: reducing the aqueous solution containing the population of the collagen microbubbles to a temperature below the gel forming temperature of the aqueous solution.

42. The method of any one of claims 25-41, wherein the aqueous solution is according to the composition of any one of claims 1-17.

43. The method of any one of claims 25-42, further comprising: applying the population of the collagen microbubbles to a wound of a patient in an effective amount to induce healing of the wound.

44. The method of any one of claims 25-42, further comprising: adding the population of collagen microbubbles to a beverage.

45. The method of any one of claims 25-42, further comprising: using the population of collagen microbubbles in tissue engineering.

46. The method of claim 45, further comprising: using the population of collagen microbubbles as a collagen scaffold.

47. The method of any one of claims 25-42, further comprising: using the population of collagen microbubbles to treat hypoxia.

48. The method of any one of claims 25-42, further comprising: using the population of collagen microbubbles as a pain remedy.

49. The method of any one of claims 25-48, wherein the fatty acid comprises stearic acid.