Patent Application
20220160752
The present invention relates to the field of biopolymers, e.g. biodegradable biopolymers, and, more specifically, to a biopolymer and related compositions, uses thereof, and methods of preparation of such biopolymer.
Biodegradable biopolymers can naturally occur as polymeric biomolecules formed during the life cycle of green plants, animals, bacteria, and fungi. Such biomolecules may typically have an advantageously high degree of biocompatibility. Animal protein-based biopolymers include, or form a substantial component of, well-known products such as wool, silk, gelatin, and collagen. Other examples include polysaccharides, such as cellulose and starch, and carbohydrate polymers produced by bacteria and fungi. Biopolymers, and particularly those of carbohydrate origin, are promising for biomedical applications in various forms, e.g. due to their advantageously low toxicity, low antigenicity, high bio-activity, biodegradability, stability, processability into complicated shapes with appropriate porosity to support cell growth, mechanical properties, and renewable nature.
The United States Congress Office of Technology Assessment classifies such biopolymers into nucleic acids, proteins, polysaccharides, polyhydroxyalkanoates and polyphenols.
Biopolymers may be generated from renewable sources and may be easily biodegradable because of the oxygen and nitrogen atoms found in their structural backbone. Biodegradation converts them to CO2, water, biomass, humid matter, and other, typically harmless, natural substances. Biopolymers can thus be naturally recycled by biological processes.
Bioactive properties of biopolymers, such as antimicrobial, immune-modulatory, cell proliferative and angiogenic properties, create a microenvironment that is favorable for the healing process and provide a plethora of applications in pharmaceutical, medical and cosmetic applications. Furthermore, one of the major advantages of biopolymers is their ability to absorb large volumes of water when in the dry state, while being able to donate water when hydrated.
The foundation of polymer science was laid with the discovery of cellulose in 1838 by the French chemist Anselme Payen, who isolated it from plant matter and determined its chemical formula, being identified as a covalently linked, high-molecular-weight macromolecule.
Cellulose is an abundant naturally occurring polymer of glucose, found as the main constituent of plants and natural fibers such as cotton and linen. Some bacteria (e.g., Acetobacter xylinum) can also synthesize cellulose. While plant cellulose is chemically identical to microbial or bacterial cellulose, it can possess a different macromolecular structure and physical properties. Natural cotton fibers and regenerated (artificial) cellulose fibers, such as viscose, modal, and lyocell, are used in many medical applications. In devices for controlled drug delivery, e.g. in the form of solid tablets, cellulose ether can be used, which allows a swelling-driven release of the drug when physiological fluids come into contact with the tablet. When the cellulose ether on the tablet surface swells, chain entanglements and a physical hydrogel are formed. As swelling proceeds from the surface to the glassy core of the tablet, the drug progressively dissolves in water and diffuses out from the polymer network.
Cellulose can also be used as scaffolds for regenerative medicine due to its excellent biocompatibility and good mechanical properties. For example, bacterial cellulose may be suitable for use in wound dressings or other wound healing applications, particularly in view of its purity and high water retention capacity.
Collagen is another great example of a biodegradable biopolymer. This is a predominant structural material in vertebrates, and the most abundant mammalian protein, e.g. accounting for about 20% to 30% of total body proteins. Collagen is synthesized by fibroblasts, which usually originate from pluripotential adventitial cells or reticulum cells. The elementary monomer molecule of collagen is rod-shaped, with a length and width of, respectively, about 3000 Å and 15 Å, and has an approximate molecular weight of 300 kDa. It is easily absorbable in the body and has a very low antigenicity. Moreover, it is non-toxic, biocompatible and biodegradable, and has a high tensile strength and high affinity with water.
In biomedical applications, collagen is used in numerous applications. For example, it can be used in tissue-based devices, such as prosthetic heart valves and vascular prostheses, which has contributed to an increased life expectancy of cardiac patients and lower risks of infection.
Collagen can also be used in drug delivery systems. However, its main application may be in collagen films used as barrier membrane. Collagen films, sheets and/or discs can be used in the treatment of tissue infections, such as infected corneal tissue or liver cancer. A soluble ophthalmic insert, in the form of a wafer or a film, is known in the art, as a drug delivery system for the treatment of infected comeal tissue using a high dose of antibiotic agents, such as gentamycin and tetracycline. Collagen films have also been used as a matrix for gene delivery carriers to promote bone formation.
Another useful medical application is the use of collagen sponges in the management of severe burns and as a dressing for severe wounds. Collagen dressings are prepared in a variety of different forms including, sponges, membrane sheets and powder, which have advantageous biological properties for such applications. In tissue engineering, collagen-based implants are widely used as vehicles for delivery of cultured skin cells and as drug carriers for skin replacement and burn wounds treatment. They have also been used as bone substitute, due to its osteoinductive activity, and as implantable carriers for bone inducing proteins, such as bone morphogenetic protein 2 (rhBMP-2).
As a haemostat, collagen achieves rapid coagulation of blood through its interaction with the platelets and can provide a temporary framework while the host cells regenerate their own fibrous stroma. The use of collagen-based haemostats has been proposed for reducing blood loss in generalized bleeding in a wide range of tissues, and for managing wounds to cellular organs, such as the liver or spleen.
Another widely used and studied biodegradable biopolymer is chitosan. Chitosan is a derivative of the natural polysaccharide, chitin. Chitin was first isolated and characterized from mushrooms, by the French chemist Henri Braconnot in 1811. It is known as the second most abundant biopolymer, i.e. it is the most abundant polysaccharide in nature after celluloses. Chitin is the main component of the exoskeleton of crustaceans and insects, and also occurs in nematodes and in the cell wall of yeast and fungi. Chitosan has an excellent biocompatibility and other advantageous properties, such as good bioactivity, biodegradability, selective permeability, polyelectrolyte action, antimicrobial activity, ability to form gel and film, chelation ability and absorptive capacity. These peculiar properties enable a wide range of useful applications of chitosan, such as a drug carrier for controlled release, an anti-bacterial, and an anti-acid. It inhibits the bacterial plaque formation and decalcification of dental enamel, promotes osteogenesis, can act as a fat absorbent and promotes the healing of ulcers and lesions.
Chitosan has an in vivo stimulatory effect on nitric oxide production and modulates peroxide production. Neutrophil activation can be inhibited by administration of chitosans having a low molecular weight and oxidation of serum albumin is commonly observed in patients undergoing hemodialysis, resulting in reduction of oxidative stress associated with uremia.
In drug delivery, chitosan may potentially improve drug absorption and stabilization of drug components to increase drug targeting. In addition, chitosan can protect DNA and increase the expression period of genes. Chitin or chitosan derivatives, which were conjugated with some kinds of anticancer agents, can execute better anticancer effects with gradual release of free drug in the cancer tissues.
As an anti-inflammatory agent, chitosan can partially inhibit the secretion of both IL-8 and TNF-α from mast cells, demonstrating that water-soluble chitosan has the potential to reduce the allergic inflammatory response. Chitosan promotes phagocytosis and production of osteopontin and leukotriene B by polymorphonuclear leukocytes, production of interleukin-1, transforming growth factor b1 and platelet-derived growth factor by macrophages, and production of interleukin-8 by fibroblasts, enhancing immune responses.
As a composite for tissue engineering, chitosan and hydroxyapatite are one of the best bioactive biomaterials in bone tissue engineering. Different types of polysaccharides from marine sources are used for the management of bone diseases, such as osteoporosis and arthritis. As a functional wound dressing, a hydrogel sheet composed of a blended powder of alginate, chitin/chitosan and fucoidan is known for creating a moist environment for rapid wound healing. It is highly biocompatible and biodegradable, has a porous structure, and is suitable for cell ingrowth, osteoconduction and intrinsic antibacterial nature. Chitosan has been developed considerably in biomedical applications, e.g. cartilage tissue engineering, wound healing and orthopedic applications. Degradable polymeric implants have the benefit that the need for a second surgical operation for removal can be avoided. Chitosan/hydroxyapatite composite materials are promising to mimic both the organic as the inorganic portion of natural bone.
Chitosan as an antimicrobial agent shows antimicrobial action in a great variety of microorganisms, including algae, fungi and bacteria. It has shown antimicrobial activity against gram-positive bacteria and various species of yeast. A chitosan gel, derived from dilution in acetic acid, was suggested as a preventive and therapeutic material for dental caries.
Hyaluronic acid is also widely distributed in the extracellular matrix and the joint liquid of mammals and has been approved for injections by the Food and Drug Administration (FDA). It is a biocompatible and biodegradable mucoadhesive polysaccharide with negative charge. The first medical application of hyaluronan for humans was as a vitreous substitution/replacement during eye surgery in the late 1950s. Hyaluronic acid polymers are used in the preparation of gels for delivery of drugs to the eye and installation into other cavities. Along with other polymers like alginic acid, HPMC (Hydroxypropyl methyl cellulose), poloxamers etc. can be used for achieving the desired property in drug delivery systems.
Comeal shields based on hyaluronic acid have demonstrated prolonged steroid delivery, with a consummate smoothing of dosage profile. Other applications of hyaluronic acid include: (i) wound healing by extracellular regeneration; (ii) epithelial regeneration; (iii) topical treatment of dry eye syndrome and Sjögren's syndrome; (iv) as a viscosity agent in pulmonary pathology for achieving alveolar patency; (v) a commercial preparation available for intra-articular injection; (vi) As a filler in rejuvenative medicine for wrinkles and cutaneous lines.
Viscosupplementation with hyaluronic acid products helps to improve the physiological environment in an osteoarthritic joint by supplementing the shock absorption and lubrication properties of osteoarthritic synovial fluid. The rationale for using viscosupplementation is to restore the protective viscoelasticity of synovial hyaluronan, decrease pain, and improve mobility. Esterified hyaluronic acid has also been used to prevent bacterial adhesion to dental implants, intraocular lenses, and catheters. Hyaluronic acid is an appropriate choice for a matrix to support dermal regeneration and augmentation. As a result of its ability to form hydrated, expanded matrices, Hyaluronic acid has also been successfully used in cosmetic applications such as soft tissue augmentation.
PDRN is another biodegradable biopolymer that possesses several activities: tissue repairing, anti-ischemic, and anti-inflammatory. These therapeutic properties may suggest a use in regenerative medicine and in diabetic foot ulcers. PDRN holds a mixture of deoxyribonucleotides with molecular weights ranging between 50 and 1500 KDa, it is derived from a controlled purification and sterilization process of Oncorhynchus mykiss (Salmon Trout), Oncorhynchus keta (Chum Salmon), or sturgeon sperm DNA. The procedure guarantees the absence of active protein and peptides that may cause immune reactions. In vitro and in vivo experiments have suggested that PDRN most relevant mechanism of action is the engagement of adenosine A2A receptors. Besides engaging the A2A receptor, PDRN offers nucleosides and nucleotides for the so called “salvage pathway.” The binding to adenosine A2A receptors is a unique property of PDRN and seems to be linked to DNA origin, molecular weight and manufacturing process. In this context, PDRN represents a new advancement in pharmacotherapy. In fact, adenosine and dipyridamole are non-selective activators of adenosine receptors and they may cause unwanted side effects; while regadenoson, the only other A2A receptor agonist available, has been approved by the FDA as a pharmacological stress agent in myocardial perfusion imaging.
PDRN has also been successfully used in cosmetic applications as a so-called injectable or topical “skin-healer”.
Biodegradable polymers such as chitosan and hyaluronic acid need to be crosslinked in order to modulate their general properties and to last long enough for delivering drugs over a desired period of time. Various chemicals have been used for crosslinking chitosan, such as glutaraldehyde, tripolyphosphate, ethylene glycol, diglycidyl ether and diisocyanate. However, the synthetic crosslinking reagents are all more or less cytotoxic and may impair the biocompatibility of a chitosan, hyaluronic acid or other biodegradable polymer delivery system. Hence, efforts were made to provide crosslinking reagents that have low cytotoxicity and that form stable and biocompatible crosslinked products.
A need exists in the art for novel biodegradable biopolymers that have a wide pleiotropic anti-inflammatory action, and good properties with respect to toxicity, antigenicity, bio-activity, biodegradability, stability, processability, mechanical properties and renewable nature. There is also a clinical need for biocompatible drug carriers that release an effective quantity of drug over a period of time for prolonged therapeutic effects. Drugs are usually admixed or entrapped physically within a polymer framework for slow drug release. Plastic polymers that are suitable as a drug carrier may, however, lack sufficient biocompatibility, or biocompatible plastic polymers may be ineffective in containing a specific drug and/or releasing the drug in an timely manner for effective therapies. Therefore, a need remains for producing biodegradable biopolymers with the beforementioned capabilities, preferably by a low-cost and efficient process and/or in a highly purified state.
It is an object of embodiments of the present invention to provide in a biopolymer, methods of producing such biopolymer and uses of such biopolymer. The biopolymer may have anti-inflammatory uses, such that such biopolymer can be used in compositions for applications in oral hygiene products, wound-care, cosmetics, pharmaceuticals, food, and/or as mucoadhesive carrier of APIs. The novel biopolymers of the present invention may be PDRN-genipin-Sacran scaffolds, PDRN-Genipin scaffolds, or Sacran-Genipin Scaffolds, or analogues thereof, wherein genipin, or an analogue or derivative thereof, functions as a natural crosslinker.
It is an advantage of embodiments of the present invention that a useful compound is provided for treatment of inflammatory gum diseases and/or as a wound healing agent.
It is an advantage of embodiments of the present invention that a biopolymer is provided that has low toxicity, low antigenicity, high bio-activity, good biodegradability, good stability, good processability into complicated shapes, e.g. with appropriate porosity to support cell growth, and/or good mechanical properties.
It is an advantage of embodiments of the present invention that genipin, analogues or derivatives thereof has good crosslinking properties.
It is an advantage of embodiments of the present invention that the provided biopolymer is renewable in nature, e.g. can be generated from renewable sources and are easily biodegradable, e.g. into harmless and/or reusable products.
In a first aspect, the present invention relates to a biopolymer comprising a polydeoxyribonucleotide (PDRN) and an anionic and ampholytic heteropolysaccharide having sulfate and carboxyl groups. The poly deoxyribonucleotide has a molecular weight in the range of 400 000 Dalton (or 400 kDa) to 3 200 000 Dalton (or 3.2 MDa), e.g. in the range of 2.0 MDa to 2.8 MDa, e.g. 2.4 MDa. The heteropolysaccharide has a molecular weight in the range of 14 000 000 Dalton (or 14 MDa) to 16 000 000 Dalton (or 16 MDa). The polydeoxyribonucleotide and the heteropolysaccharide are crosslinked by genipin, a water soluble aglycon, or an analogue or derivative thereof.
PDRN may advantageously have pharmacological activity without transmitting genetic information, e.g. unlike DNA and/or RNA fragments. PDRN may refer to pure PDRN, or an active form of 95% or higher purity. PDRN may be obtained from sturgeon, salmon (Oncorhynchus keta), or trout (Oncorhynchus mykiss) semen, e.g. by subjecting the material to a high heat treatment. The INCI name is Sodium DNA. For example, PDRN may be a sodium DNA extracted from semen or testis tissue. PDRN may be derived from any one or more of fish, animals, and plants. Specifically, the PDRN may be derived from sperm, semen and/or testes of, e.g. preferably, fish. PDRN may be composed of a DNA polymer, which may promote DNA synthesis and rapid tissue regeneration. For example, PDRN may be anti-inflammatory and may shorten wound healing time and/or activate cell regeneration. PDRN may activate the production of proteins and promote the differentiation of cells. The G-C content (%) of PDRN may be about 40%. PDRN may also comprise a mixture of DNA and/or RNA fragments. PDRN in the context of the present disclosure may also refer to derivatives or analogues of PDRN.
A biopolymer in accordance with embodiments of the present invention may be a biopolymer as obtainable by a method in accordance with embodiments of the present invention.
In a polymer in accordance with embodiments of the present invention, the PDRN may be derived from the testes or milt of Onchorhynchus species.
In a biopolymer in accordance with embodiments of the present invention, the heteropolysaccharide may be sacran.
Sacran may refer to a sulfated polysaccharide, e.g. in accordance with the CAS Registry Number 1039552-36-7. Sacran may have a repeat structure of a sugar chain unit where a sugar constituent having a hexose structure and a sugar constituent having a pentose structure are conjugated together in a linear chain or a branched chain through an α-glycoside bond or a β-glycoside bond. The sugar chain unit may contain a lactated, sulfated sugar as the sugar constituent. In the sugar chain unit, 2.7 or more hydroxyl groups per 100 hydroxyl groups may be sulfated or sulfur elements may occupy 1.5% by weight or more of all of the elements.
In a biopolymer in accordance with embodiments of the present invention, the sacran may be obtained from Aphanothece sacrum. For example, sacran may be obtained by protonating a polysaccharide extracted from Aphanothece sacrum, and by performing partial hydrolysis thereon. However, embodiments are not necessarily limited to sacran obtained (derived) from Aphanothece sacrum.
Ginipin may refer to methyl (1R,2R,6S)-2-hydroxy-9-(hydroxymethyl)-3-oxabicyclo[4.3.0]nona-4,8-diene-5-carboxylate. Ginipin is an aglycone derived from geniposide, an iridoid glycoside, which may be found in fruit of Gardenia jasminoides. Genipin can be prepared by oxidation followed by reduction and hydrolysis or by enzymatic hydrolysis of the parent compound geniposide, or, alternatively, racemic genipin can be prepared synthetically.
The term “genipin or an analogue or derivative thereof” refers to genipin, its derivatives, analogs and any stereoisomers or mixtures of stereoisomers of genipin. Thus, genipin, its derivatives, analogs, any stereoisomers, or mixtures of stereoisomers of genipin, or any combination thereof, can be used as the cross-linker referred to in embodiments of the present invention. Illustrative genipin analogues and derivatives include, but are not limited to, a compound represented by:
wherein R1 represents lower alkyl, benzyl, or C1-C11 alkylcarbonyl which can be substituted with phenyl, phenoxy, pyridyl, t-butyl or thienyl, R2 represents hydroxymethyl, formyl, acetyl, hydroxyiminomethyl, methoxyiminomethyl, lower alkylaminomethyl, acetylthiomethyl, mercaptomethyl, 2,2-dimethyl-1,3-dioxolan-5-ylmethyloxymethyl, 2,3-dihydroxypropyloxymethyl, 6-aminofuran-9-ylmethyl, 4-amino-2-hydroxy-5-methylpyrimidin-1-ylmethyl, 2,4-dihydroxy-5-methylpyrimidin-1-ylmethyl, 5-hydroxymethyl-1,3-oxathiolan-2-yl, or C1-C11 alkylcarbonyloxymethyl which can be substituted with phenyl, phenoxy, pyridyl, t-butyl or thienyl, and R3 represents methoxycarbonyl, formyl, hydroxyiminomethyl, methoxyiminomethyl, 4-methoxybenzyloxymethyl or acetyloxymethyl, provided that R3 is not methoxycarbonyl when R1 is lower alkyl, acetyl or t-butylcarbonyl and R2 is hydroxymethyl, formyl, hydroxyiminomethyl, acetyloxymethyl or t-butylcarbonyloxymethyl, and R3 is not acetyloxymethyl when R1 is t-butylcarbonyl and R2 is acetyloxymethyl, its pharmaceutically acceptable salt, or stereoisomer.
Another illustrative genipin analogue or derivative may be represented by the following formula:
in which R1 represents lower alkyl; R2 represents lower alkyl, pyridylcarbonyl, benzyl or benzoyl; R3 represents formyl, hydroxymethyl, azidomethyl, 1-hydroxyethyl, acetyl, methyl, hydroxy, pyridylcarbonyl, cyclopropyl, aminomethyl substituted or unsubstituted by (1,3-benzodioxolan-5-yl)carbonyl or 3,4,5-trimethoxybenzoyl, 1,3-benzodioxolan-5-yl, ureidomethyl substituted or unsubstituted by 3,4,5-trimethoxyphenyl or 2-chloro-6-methyl-3-pyridyl, thiomethyl substituted or unsubstituted by acetyl or 2-acetylamino2-ethoxycarbonyethyl, oxymethyl substituted or unsubstituted by benzoyl, pyridylcarbonyl or 3,4,5-trimethoxybenzoyl; provided that R3 is not methyl formyl, hydroxymethyl, acetyl, methylaminomethyl, acetylthiomethyl, benzoyloxymethyl or pyridylcarbonyloxymethyl when R1 is methyl, and its pharmaceutically acceptable salts, or stereoisomers.
Another illustrative genipin analogue or derivative may be represented by the following formula:
wherein R4 represents lower alkoxy, benzyloxy, benzoyloxy, phenylthio, C1˜C12 alkanyloxy substituted or unsubstituted by t-butyl, phenyl, phenoxy, pyridyl or thienyl; R5 represents methoxycarbonyl, formyl, hydroxyiminomethyl, methoxyimino-methyl, hydroxymethyl, phenylthiomethyl or acetylthiomethyl; provided that R5 is not methoxycarbonyl when R4 is acetyloxy; and its pharmaceutically acceptable salts, or stereoisomers.
Another illustrative genipin analogue or derivative may be represented by
where R6 represents hydrogen atom, lower alkyl or alkalimetal; R7 represents lower alkyl or benzyl; R8 represents hydrogen atom or lower alkyl; R9 represents hydroxy, lower alkoxy, benzyloxy, nicotinoyloxy, isonicotinoyloxy, 2-pyridylmethoxy or hydroxycarbonylmethoxy; provided that R9 is not hydroxy or methoxy when R6 is methyl and R8 is hydrogen atom; and its pharmaceutically acceptable salts, or stereoisomers.
Another illustrative genipin analogue or derivative may be represented by
wherein R10 represents lower alkyl; R11 represents lower alkyl or benzyl; R12 represents lower alkyl, pyridyl substituted or unsubstituted by halogen, pyridylamino substituted or unsubstituted by lower alkyl or halogen, 1,3-benzodioxolanyl; R13 and R14 each independently represent a hydrogen atom or join together to form isopropylidene; and its pharmaceutically acceptable salts, or stereoisomers.
Other illustrative genipin analogues and/or derivatives are disclosed in U.S. Pat. No. 7,649,014, the content of which is incorporated herein by reference.
In a second aspect, the present invention relates to a method of producing a biopolymer, e.g. a biopolymer in accordance with embodiments of the first aspect of the present invention. The method comprises providing a polydeoxyribonucleotide (PDRN), or a derivative or modification thereof, having a molecular weight in the range of 400 000 Dalton (or 400 kDa) to 3 200 000 Dalton (or 3.2 MDa). The method comprises providing an anionic and ampholytic heteropolysaccharide having sulfate and carboxyl groups and a molecular weight in the range of 14 000 000 Dalton (or 14 MDa) and 16 000 000 Dalton (or 16 MDa). The method comprises crosslinking the polydeoxyribonucleotide and the heteropolysaccharide by genipin, a water soluble aglycon, or an analogue or derivative thereof.
A method in accordance with embodiments of the present invention may comprise dissolving PDRN matrix in deionized water to obtain a first solution of PDRN in the range of 0.005 wt % to 10 wt %.
A method in accordance with embodiments of the present invention may comprise dissolving Sacran matrix in deionized water to obtain a second solution of Sacran in the range of 0.005 wt % to 10 wt %.
A method in accordance with embodiments of the present invention may comprise adding Genipin into a blended solution of said first solution and said second solution to obtain a solution of Genipin in the range of 0.05 wt % to 2.5 wt %, preferably 2.5 wt %. Thus a third solution of Genipin, Sacran and PDRN may be obtained by this blending.
In a method in accordance with embodiments of the present invention, the blending may comprise continuous stirring at a temperature in the range of 40° C. to 50° C., e.g. 45° C., for at least 4 hours, e.g. 8 h, or even longer, e.g. in the range of 6 hours to 24 hours.
A method in accordance with embodiments of the present invention may comprise dispersing the (third) solution of Genipin, PDRN and Sacran under magnetic stirring.
A method in accordance with embodiments of the present invention may comprise filtrating said (third) solution to eliminate air bubbles, e.g. after said step of dispersing.
A method in accordance with embodiments of the present invention may comprise crosslinking the (third) solution, e.g. in an oven, at a temperature in the range of 30° C. to 45° C. for at least 12 hours, e.g. preferably in the range of 35° C. to 40° C., e.g. 37° C. Preferably, the crosslinking step comprises a heating step, e.g; at a substantially constant temperature, for at least 24 hours, e.g. 24 hours, 30 hours, 36 hours or even 48 hours.
A method in accordance with embodiments of the present invention may comprise gelling the crosslinked solution to obtain a gel comprising biopolymer scaffolds. For example, the crosslinked solution may be gelled at a temperature in the range of 0° C. to 10° C., preferably in the range of 2° C. to 6° C., e.g. 4° C., for 24 hours or longer.
A method in accordance with embodiments of the present invention may comprise lyophilizing the scaffolds.
Lyophilizing the scaffolds may comprise freezing the gel at a temperature, e.g. in the range of −40° C. to −100° C., e.g. in the range of −70° C. to −90° C., e.g. −80° C., for at least 12 hours, e.g. 24 hours or even longer, and lyophilizing the frozen scaffolds e.g. for at least 12 hours, preferably at least 24 hours, e.g. for 48 hours or even longer.
In a method in accordance with embodiments of the present invention, the (third) solution may comprise a PDRN to Sacran ratio in the range of 70:30 to 30:70, preferably 60:40, e.g. 70:30, 60:40, 50:50, 40:60 or 30:70 (embodiments including any intermediate value).
In a third aspect, the present invention relates to a composition comprising the biopolymer in accordance with embodiments of the first aspect of the present invention for use in a biomedical, cosmetic, food or agricultural product.
In a composition in accordance with embodiments of the present invention, the biopolymer may be used as a carrier for direct or sustained release of an active pharmaceutical ingredient.
In a composition in accordance with embodiments of the present invention, the active pharmaceutical ingredient may be an epidermal growth factor.
In a fourth aspect, the present invention relates to a pharmaceutical comprising the biopolymer in accordance with embodiments of the first aspect of the present invention for treating inflammation and/or impaired wound healing.
In a fifth aspect, the present invention relates to the use of a composition in accordance with embodiments of the third aspect of the present invention or the pharmaceutical in accordance with embodiments of the fourth aspect of the present invention for the treatment of inflammation and/or impaired wound healing, e.g. for the treatment of a skin inflammatory disease.
The independent and dependent claims describe specific and preferred features of the invention. Features of the dependent claims can be combined with features of the independent claims and with features of other dependent claims as deemed appropriate, and not necessarily only as explicitly stated in the claims.
FIGS. TA-F show FT-IR spectroscopy analysis of FIG. TA PDRN,
The drawings are schematic and not limiting. Elements in the drawings are not necessarily represented on scale. The present invention is not necessarily limited to the specific embodiments of the present invention as shown in the drawings.
Notwithstanding the exemplary embodiments described hereinbelow, is the present invention only limited by the attached claims. The attached claims are hereby explicitly incorporated in this detailed description, in which each claim, and each combination of claims as allowed for by the dependency structure defined by the claims, forms a separate embodiment of the present invention.
The word “comprise,” as used in the claims, is not limited to the features, elements or steps as described thereafter, and does not exclude additional features, elements or steps. This therefore specifies the presence of the mentioned features without excluding a further presence or addition of one or more features.
The embodiments of the present invention are specifically described below with reference to the embodiments, so as to facilitate the understanding of the present invention by those skilled in the art. It should be noted that the embodiments are only used for further explanation of the present invention, cannot be understood to limit the protection scope of the present invention, and a person skilled in the art will recognize that the protection scope of the present invention can be better understood by those skilled in the art, the non-essential improvement and adjustment made by the method disclosed by the invention should still be within the protection scope of the invention.
The raw materials are commercially available products; the process steps or the preparation method which are also not always described in detail, are process steps or preparation methods which are all known by those skilled in the art. Meanwhile, raw materials being used are not always described in detail.
In this detailed description, various specific details are presented. Embodiments of the present invention can be carried out without these specific details. Furthermore, well-known features, elements and/or steps are not necessarily described in detail for the sake of clarity and conciseness of the present disclosure.
The present invention relates to a novel engineered biodegradable biopolymer. It comprises the crosslinking of two biodegradable biopolymers: the first being PDRN, or a derivative or modification thereof, the other being Sacran, or a derivative or modification thereof, with Genipin functioning as a natural crosslinking agent.
Other possible formations from crosslinking Sacran to another moiety (such as, but not limited to, hyaluronic acid, chitosan, cellulose, heparin, collagen, elastin, heparosan, carrageenan, starches, alginate, gelatin, or derivatives thereof), or by using another synthetic, or natural crosslinking agents (for example, but not limited to, glutaraldehyde, tripolyphosphate, ethylene glycol, diglycidyl ether and diisocyanate, Proanthocyanidin, Chitosan, Epigallocatechin-3-gallate, Low dose riboflavin/UVA-activated riboflavin, hesperidin, Galardin, or derivatives thereof) are also within the scope of this present invention.
In accordance with embodiments of the present invention, there is provided the process of obtaining the Genipin-crosslinked PDRN/Sacran scaffolds, which have shown to exhibit many of the desired characteristics important for optimal therapeutic function. In particular, the Genipin-crosslinked PRDN-Sacran scaffolds with strong muco-adhesive, anti-inflammatory, angiogenic, and drug loading capability may be suitable in therapeutic applications that involve many inflammatory diseases and tissue engineering applications.
Also in accordance with embodiments of the present invention, is the application of the Genipin-crosslinked PDRN/sacran scaffolds serving as hydrogel implants or in the form of dressings for improved wound healing/closure. Such implants or dressings can also be loaded with APIs in order to improve the healing rate of the wound. The particular use of EGF in combination with the Genipin-crosslinked PDRN/Sacran scaffolds, Genipin-crosslinked PDRN scaffolds, or Genipin-crosslinked Sacran scaffolds, is also subject to present invention and further explained in detail further hereunder. The use of any other API in combination with the Genipin-crosslinked PDRN/Sacran scaffolds, Genipin-crosslinked PDRN scaffolds, or Genipin-crosslinked Sacran scaffolds, is also subject to present invention.
The use of PDRN-Genipin-Sacran in biomedical, cosmetic, food, and agricultural applications are also within the scope of this invention.
The use of PDRN and Sacran combined in a mixture, without being crosslinked, in all biomedical, cosmetic, food, and agricultural applications are also within the scope of this invention.
The use of PDRN-Genipin crosslinked to any other biological material, in all biomedical, cosmetic, food, and agricultural applications are also within the scope of this invention.
The use of Sacran-Genipin crosslinked to any other biological material, in all biomedical, cosmetic, food, and agricultural applications are also within the scope of this invention.
The use of PDRN-Genipin-Sacran crosslinked to any other biological material, in all biomedical, cosmetic, food, and agricultural applications are also within the scope of this invention.
According to embodiments of the present invention, the PDRN moiety of the PDRN-Genipin-Sacran conjugated polymer may be a mixture of highly polymerized deoxyribonucleotides or polydeoxyribonucleotides (hererinafter referred to as “PDRN”). PDRN in preferably in the form of a sodium salt by which non-denaturing techniques ensures a perfect protection of the molecular structure of the DNA (in particular preserves the DNA in its double-stranded form), and thus preserves its physiological activity. Other preferable forms may include iron, chromium, magnesium, and manganese salts. Preferably, the highly polymerized DNA of the composition (PDRN) of the invention may be obtained by the implementation of non-denaturing processes. PDRN is known for its tissue regeneration activity at cellular level through the stimulation of the production of white blood cells, lymphocytes and neutrophils. It may be obtained from naturally-occurring sources such as animal sources, and in particular from the reproduction cells of certain aquatic animals, particularly such as the testes, milt or roe sacs of rainbow/steelhead trout (Oncorhynchus mykiss). Other species also include: cutthroat trout (Oncorhynchus clarki), pink salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), coho salmon (Oncorhynchus kisutch), masu/cherry salmon (Oncorhynchus masou), sockeye/kokanee (Oncorhynchus nerka), and chinook salmon (Oncorhynchus tshawytscha). Although the milt from acispencer, huso, scaphirhynchus, and pseudoscapirhynchus sturgeon species can also be used, due to them being critically endangered, their use may be less preferred.
PDRN may be extracted and purified at high temperature, a procedure that allows to recover a >95% pure active substance with inactivated proteins and peptides. This latter may contribute to the safety of the product and avoid immunological side effects. Spermatozoa may be preferable as source material to provide highly purified DNA without risk of impurity such as peptides, proteins and lipids which can remain from the somatic cells.
Such process may relate to extraction and purification under high temperature: I) enzyme lysis of the protein organic matrix; II) clarifying filtration of the resulting solution; III) precipitation with quaternary ammonium salts; IV) decomplexing of the precipitate; V) molecular selection using column chromatography; VI) precipitation of the eluate using alcohol until a negative protein reaction is indicated in the eluate, and VII) recovering of the purified biological material in the eluate. “Highly polymerized DNA” may refer to a DNA having a molecular mass of between 1000 and 5000 kDa, preferably between 1500 and 4000 kDa, preferably between 1750 and 3000 kDa, and preferably of 2400 kDa.
The nucleotide composition of the DNA extracted from rainbow/steelhead trout (Oncorhynchus mykiss) testes is as follows:
Adenine: 29.7±0.08
Thymine: 29.1±0.09
Guanine: 20.8±0.08
Cytosine 20.4±0.07
A native DNA sodium salt may be used in accordance with embodiments of the invention; e.g. having a specific molecular weight between 200×104 daltons to 240×104 daltons, e.g. and having a hyperchromicity effect (G): G 41%.
As an example, 0.20 g of the preparation may be placed into a measured 30 ml retort, 0.1% sodium chloride solution may be added, the preparation may be dissolved and the volume may be brought up to the mark. 1 ml of the solution may be placed into a measured 250 ml retort, the volume may be brought up to the mark with 0.2% sodium chloride solution. Absorption of the received solution (D260) may be measured.
10 ml of solution may be placed into a retort with thin section, 10 ml of 15% chloric acid solution, closed and boiled on a water bath for 25 min. The solution may be cooled own to the normal temperature, transferred by quantity into a measured 200 ml retort, and the volume may be brought up to the mark with water. 10 ml of this solution may be placed into a measured 100 ml retort and the solution volume may be brought up to the mark with water. Absorption of the received solution (D1260) may be measured.
The PDRN moiety of the total PDRN-Genipin-Sacran may suggest a wider pleiotropic modality and anti-inflammatory action versus PDRN in its native state, due to the crosslinking with highly ampholytic sulfated Sacran. Indeed, the studies performed by the authors of the present invention show a significant reduction of the serum levels of a number of inflammatory mediators: Th-1 and Th-2 cytokines such as Interferon-gamma (IFN-γ), Tumor necrosis factor alpha (TNF-α) (although also downregulated by PDRN), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Eotaxin, and Monocyte chemoattractant protein-1 (MCP-1).
In vitro and in vivo experiments have suggested that PDRN most relevant mechanism of action is the engagement of adenosine A2A receptors, which are responsible for sending out a regeneration signal to secrete various growth factors. It is known as a component that promotes capillary production by stimulation of vascular endothelial growth factor (VEGF), thus improving blood circulation, anti-inflammatory action and prevention of capillary leakage. By stimulating an even wider pleiotropic pathway, PDRN increases anti-inflammatory Interleukin-10 factor (IL-10), and decreases pro-inflammatory cytokines such as Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-a), and High mobility group protein 1 (HMGB1) which are suggested to contribute to the pathogenesis of various chronic inflammatory and autoimmune diseases, and cancer. High serum levels are found in several inflammatory events including but not limited to; sepsis, periodontitis, gingivitis rheumatoid arthritis, artherosclerosis, chronic kidney disease, systemic lupus erythematosus (SLE). Furthermore PDRN positively stimulates the secretion of Epidermal growth factor, Fibroblast growth factor, and Insulin growth factor, which are key mediators in fibroblast, osteoblast, and chondrocyte proliferation, and differentiation.
Besides engaging the A2A receptor, PDRN also provides nucleosides and nucleotides for the so-called “salvage pathway.” The binding to adenosine A2A receptors however, is a unique property of PDRN and seems to be linked to DNA origin, molecular weight and manufacturing process. In this context, PDRN represents a new advancement in pharmacotherapy and may be particularly useful towards creating modified biodegradable biopolymers with improved pharmacological action.
In terms of the chemical structure, PDRN may be composed of low-molecular weight DNA and may be a linear polymer comprising 50-2,000 base pairs. PDRN is degraded by active membrane enzymes to serve as a source of purine and pyrimidine deoxynucleosides and deoxyribonucleotides, which increase cellular proliferation and activity in different tissues. The structure of an illustrative polydeoxyribonucleotide (PDRN) segment held by phosphodiester bonds is shown hereunder:
Genipin may be a preferred water-soluble bi-functional natural crosslinking agent of choice. This Genipin may relate to a 226 Da aglycone derived from geniposide, an iridoid glucoside. Geniposide is present in the fruit of Gardenia jasminoides, a common flower in many parts of Asia. It is widely used in herbal medicine, and the dark blue pigments obtained by its spontaneous reaction with amino acids or proteins have been used in the fabrication of food dyes. Other applications involve the preparation of gelatin capsules and the immobilization of enzymes. At acidic and neutral pH, Genipin reacts with primary and tertiary amines of biopolymers and forms mono- up to tetramer crosslinks. With increasingly basic conditions, Genipin further undergoes ring-opening self-polymerization with increasing polymer length prior to binding to primary or tertiary amines. With increasing polymer length (˜4-88-mers), amine reactions with Genipin slows, which leads to less reduced enzyme digestibility and swelling by Genipin. The feasibility and benefit of employing Genipin as an alternative (Genipin has a low acute toxicity, with LD50 i.v. 382 mg/k in mice) to higher toxicity crosslinkers like glutaraldehyde has been demonstrated in a range of applications, including heart valves, pericardial patches, conduits for nerve growth guidance, scaffolds for tissue-engineered cartilage, and decellularized tracheal transplantation, and as a more general application to augment the strength and degradation properties of collagen-based gels. The authors of the present invention have found that genipin can be used as a suitable crosslinking agent for the formation of PDRN-Sacran hydrogels.
An illustrative structure of Genipin is presented hereunder:
Although the structure presented above shows the natural configuration of genipin, any stereoisomer or mixture of stereoisomers of genipin as shown later may be used as a crosslinking reagent, in accordance with the present invention.
Sacran may refer to an anionic and ampholytic heteropolysaccharide with sulfate and carboxyl groups. It may be extracted from the Japanese indigenous cyanobacterium Aphanothece sacrum, which is mass-aquacultured in rivers with a high ionic concentration and possesses plenty of jelly-like extracellular matrix with high water content (97.5-98.3%). Its structure can be very complex due to many kinds of sugar residues (galactose, glucose, mannose, xylose, rhamnose, fucose, galacturonic acid, and glucuronic acid) and may contain traces of alaline, galactosamine, uronic acids (which uronic acids have yet to be determined chemically), and muramic acid; 11% of its monosaccharides may contain a sulfate group of which 22% may contain a carboxyl group. That is, the aqueous solution of sacran is a polyelectrolyte.
Due to its electric charges on the chain, sacran changes its conformation depending on the sacran concentration, e.g., helix transition concentration, where the sacran chain change from a random coil to double helix occurs at 0.09 wt. % and gelation concentration, where sacran shows a transition from liquid to weak gel at 0.25 wt. %.
Sacran shows various unique phenomena such as high swelling and anisotropic swelling behaviors, liquid crystalline (LC) behavior, ion adsorption behavior, and anisotropic diffusion behavior in highly ordered sacran films. The retention capacity for uncross-linked sacran exceeds higher than 6000 mL/g for pure water and 2700 mL/g for NaCl aqueous solution. Similar high swelling behaviors were also found for cross-linked sacran hydrogels (e.g., 6100 mL/g for pure water and 530 mL/g for 0.9 wt. % for saline solution).
These high-swelling behaviors might originate from the large mesh consisting of extremely long chains of sacran or macroscopic LC domains with millimeter to several centimeter scales in which sacran chains are aligned by self-organization. For ion adsorption, sacran shows a feature that the adsorption ratio for sacran against trivalent neodymium ions (Nd3+) is twice that for sodium alginate.
Thus, sacran demonstrates various unique phenomena originating from the feature of super megamolecules. Anomalous property can also be seen in the rheological properties of sacran; that is, the viscosity increases over time when a steady shear with low shear rates is applied.
An illustrative structure of sacran chains, where the sequences in monosaccharide triads, pattern of glycoside linkages, steric conformations of hydroxyls or other substitutes are tentative, is pictured hereunder:
Crosslinking Reaction
The reaction mechanism is a two-step process requiring two distinct reactions in an aqueous solution. The first reaction results in the heterocyclic linking of Genipin and occurs when the ring-opening reaction of Genipin undergoes a nucleophilic attack by the carbonyl & amine groups on the purine and pyrimidine rings of Adenine and Guanine (as purines), and Cytosine (as pyrimidine) in PDRN; and the many carboxyl groups present in Sacran. The second, slower, reaction occurs when the ester group on genipin undergoes a nucleophilic substitution of the ester group of genipin to form a secondary carbonyl and amide link with PDRN and carboxyl link with Sacran.
The resultant covalent crosslinks between the primary and secondary amine residues leave very minimal residual toxicity. In addition, the resultant crosslinked material can form ionic complexes with charged molecules and therefore may aid in sequestering proteins as well.
An illustrative genipin cross-linked PDRN-Sacran hydrogel may comprise the following raw materials in percentage by weight: 0.05 wt %-2.5 wt % of genipin, 1 wt %-10 wt % of matrix (PDRN-Sacran) and the balance of water. The composition of the matrix can be divided into the following ratios (for example): PDRN 70: Sacran 30, PDRN 60: Sacran 40, PDRN 50: Sacran 50, PDRN 40: Sacran 60, PDRN 30: Sacran 70. By carrying out crosslinking by virtue of optimized genipin concentration, the obtained biogel has the properties of low toxicity, good water-absorbing property and mechanical property and the like. The resulted ratio in examples relating to the present invention is PDRN 60: Sacran 40, yet is merely illustrative and functions merely as an example of many variations in composition in accordance with embodiments.
Example of Preparation of PDRN-Genipin-Sacran Crosslinked Biogel
As an example, 0.05 grams of PDRN was dissolved in deionized water (100 ml), while 0.005 grams of Sacran was dissolved in deionized water (100 ml). Both solutions were blended together under continuous stirring at 45° C. for 8 h. Different concentrations of genipin (0.5% or 2.5% (w/w)) were added into the mixture and dispersed with magnetic stirring. The solutions were filtrated twice to eliminate air bubbles and poured into 35 mm Petri dishes, which were put in a constant temperature oven at 37° C. to crosslink for 24 h, after that in a freezer at 4° C. to gel for 24 h, followed by an ultra cold freezer at −80° C. to pre-freeze for 24 h respectively. The frozen scaffolds were lyophilized for 48 h. Non-crosslinked PDRN/Sacran scaffolds were prepared without genipin using the same method. The scaffolds were balanced with Na2HPO4 solution (0.1M) and rinsed for 24 h with deionized water. Finally, the scaffolds were pre-freezing at −80° C. for 24 h and then lyophilizing for 48 h.
As a further control group, the PDRN-Sacran scaffolds crosslinked by glutaraldehyde and EDC were prepared according to methods known in the art. Glutaraldehyde is the most commonly used synthetic crosslinker, but it is poisonous and seriously impairs biocompatibility of crosslinked materials. EDC is a water soluble and non-toxic crosslinker, but the degree of crosslinking and uniformity is rather low.
The physico-chemical properties of the blends were analyzed (using thermogravimetric analysis, TGA; Fourier transform infrared-attenuated total reflectance spectroscopy, FTIR-ATR; scanning electron microscopy, SEM), as well as the functional characteristics in view of their potential application in tissue engineering (mouse fibroblast culture tests). Mechanical properties (stress-strain and creep tests) of crosslinked samples were also measured in a dry state by means of an apparatus based on an isotonic force transducer. Efficacy of genipin-crosslinking was evaluated by means of swelling and dissolution measurements carried out in media simulating physiological conditions. Fibroblasts adhesion tests performed on crosslinked cast films showed the biocompatibility of genipin-crosslinked samples.
Experimental Results
FT-IR spectra were recorded on a SmartSpec 3000 spectrophotometer (Bio-Rad) in the wave number range 4000-400 cm−1 using KBr pellets.
The scaffold samples (a total of 2% w/w) were mixed with potassium bromide. The mixtures were then homogenized into powders using a BeadBug 6 Position Homogenizer (Thomas scientific). The disks were compressed for scanning. At a minimum, each sample was assessed in triplicate. For SEM micrograph analysis, the scaffolds were cut into small identical samples using a surgical knife blade. The samples were all attached to aluminum stubs with double-sided sticky tabs while coated with gold for their analysis. With a JSM-F100 Field Emission Scanning Electron Microscope (Jeol). The pore dimensions of the genipin, glutaraldehyde and EDC crosslinked PDRN/Sacran scaffolds were measured by ImageJ software.
The FT-IR spectroscopy analysis in
The morphology of the scaffolds which were crosslinked by different crosslinkers is shown by SEM micrograph analysis in
A thermogravimetric analysis was performed using a TGA 5500 thermogravimetic analyzer (TA instruments) where thermal degradation was measured under nitrogen atmosphere. The experiments were performed at a 5° C./min heating rate in the 40-750° C. temperature range.
Two weight loss phenomena are displayed as decomposition traces of both crosslinked PDRN/Sacran, and uncrosslinked but blended PDRN/Sacran samples show water evaporation at 50-150° C. and polymer pyrolysis in the 200-500° C. temperature range. The maximum degradation rate temperatures (TdS) of all samples derived from the corresponding PDRN/Sacran 60/40 samples are shown in the table hereinbelow.
The Sacran sample is more stable than the PDRN sample, both in a crosslinked an uncrosslinked state. The DTG curves in
Swelling and dissolution tests were performed on uncrosslinked and crosslinked PDRN/Sacran film samples (measurements: 2×2 cm2). The film samples were weighed and then put in phosphate buffered saline solution (Gibco PBS, Thermo Fisher) with pH 7.4 at 37.5° C. At various times (1, 3, 6, 24 and 48 h), the swollen film samples were taken out of the PBS solution and dried superficially by contact with filter paper and weighed again.
The swelling percentage was calculated as % Sw, =[(Ws−Wi)/Wi]×100, where Ws and Wi are the film sample weights before and after swelling, respectively. Samples were weighed again after drying them at 37.5° C. for 48 hours in a vented oven.
Solubility percentages were calculated as % S=[(Wi−Wd)/Wi]×100, where Wd is the dried film sample weight after dissolution testing. Each test consisted of measurements in three replicates and the results were expressed as average values.
By increasing the PDRN amount, the stability of PDRN/Sacran samples in aqueous media increases, due to the lesser solubility of PDRN in media having a higher pH than 6.5. The differences between the degrees of PDRN and PDRN/Sacran 60/40 are not statistically significant after 24-48 h (p>0.05).
The swelling and dissolution degrees of the crosslinked Sacran film samples as function of the Genipin amount after 24 h are shown in
This result confirms that the water stability of Sacran (and therefore also its crosslinking degree) does not vary appreciably using higher Genipin contents than 2.5% (w/w).
The swelling and dissolution degrees of the crosslinked PDRN/Sacran film samples after 24 h, respectively are shown in
These findings can be attributed to the prevalence of oligomerisation reactions of Genipin on pure PDRN, due to the more acidic characteristics of the reaction environment. A higher degree of Genipin oligomerisation leads to a porous network with higher swelling properties. Independently from the Genipin amount, the dissolution degree of crosslinked PDRN film samples are close to 0 wt. % after 24 h. By increasing the Sacran amount, the dissolution degrees for PDRN/Sacran—0.5 Genipin increase, and are found to be much higher than those of the corresponding compositions crosslinked with 2.5 (w/w) Genipin (p<0.05). With respect to the film samples crosslinked with 0.5 (w/w) Genipin, the dissolution degree of the PDRN/Sacran—2.5 GP film samples have a lower dependence on the Sacran amount. The various differences between the dissolution degrees of PDRN/Sacran 60/40—2.5 Genipin, PDRN/Sacran 20/80—2.5 Genipin and Sacran—2.5 Genipin are not statistically significant (p>0.05). In fact, Sacran—2.5 Genipin only loses 14.6 wt. % after 24 h immersion in PBS.
These results conclude that crosslinking treatment using 2.0 to 2.5 wt % Genipin allows the obtainment of film samples with higher water stability.
To measure the tensile properties of the Genipin-crosslinked PDRN/Sacran films, an isotonic transducer was used with an applied force at 1 mN resolution.
The tensile properties of GP-crosslinked CS/G films were measured using an isotonic transducer (model MLT0015, Harvard Apparatus), where the applied force has a resolution of 1 mN. The test specimens were dry cast films with 2 cm in length, 1 cm in width and a height of around 150 μm. An optical microscope (Bioscience 40-1000× Trino, Bresser) equipped with a digital camera, was used to record images of each scaffold and to obtain the correlating data on initial dimensions and geometry. Along the length of the film samples, traction force was applied. For each composition, three specimens were tested. From the slope of the initial linear portion of the stress-strain curve, the Young's modulus of each sample was calculated. On identical film samples, creep measurements were performed, using the same instrument as for the tensile tests. The load corresponding to 10% of the maximum elastic deformation of the specimens was “instantaneously” applied to each film samples and then kept constant for a period of 1 h, during the developing strain γ(t) was measured. The recoverable strain was monitored for further 60 min (creep recovery measurement) after load removal.
The elastic moduli of the Genipin-crosslinked film samples in a dry state, as a function of Sacran content and Genipin amount are shown in
The differences between the elastic modulus of the film samples crosslinked with 0.5 wt % and 2.5 wt % Genipin amounts, however, were only statistically significant for samples that contain 80 wt % Sacran. For blend crosslinked with the same Genipin amount, on the other hand, the elastic modulus decreases with increasing the Sacran content. The creep and creep-recovery curves for PDRN/Sacran—0.5 Genipin film samples normalised with respect to the applied stress, i.e. compliance curves as exemplary of the creep and creep-recovery behavior of Genipin-crosslinked film samples, are shown in
On cast films, cell adhesion and proliferation tests were performed, using NIH-3T3 mouse fibroblast cells. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher) with high glucose, 15% fetal bovine serum (FBS, Sigma-Aldrich), 2% glutamine (Sigma-Aldrich), 250 U/mL of Penicillin (Sigma-Aldrich) and 200 μg/mL of streptomycin (Sigma-Aldrich). 120,000 cells/mL were the typical seeding densities. The film samples were placed in a 24-well plate (Thermo Fisher), after a twice washing and sterilization cycle. The film samples were precoated with 0.1 wt % poly-l-lysine in phosphate buffered saline (Sigma-Aldrich). To each well, 1.5 ml of adhesion factor solution was added and left in incubation for 1 h at 37.5° C. and finally aspired. Cell adhesion measurement was performed on three film samples for each time (4, 24, 48 h). The culture medium was removed and substrates with attached cells were rinsed with phosphate buffered saline (Sigma-Aldrich) after each culture time. The attached cells were fixed by addition of 4.5% (v/v) formaldehyde (Sigma-Aldrich) solution in phosphate buffered saline for 10 min. and then stained with Coomassie Blue (Thermo Fisher) solution for 10 min. Optical microscopy (Bioscience 40-1000× Trino, Bresser) was used to analyze the film samples for the ratio between the number of cells on the polymeric structures, and as an index of cell density, the total area of the polymer substrate was calculated. For comparison to evaluate the effect of scaffold topography on cell proliferation, both cells of a reference sample and cells seeded onto the scaffolds were examined. Poly-1-lysine was selected as a control, respectively for the test on mouse fibroblasts.
The crosslinked samples were found to be biocompatible and support cell adhesion and proliferation particularly well. As such, embodiments of the present invention are considered to be particularly suitable for use as scaffolds in tissue engineering.
Blank Genipin-Crosslinked PDRN/Sacran and EGF-Loaded Genipin-Crosslinked PDRN/Sacran Hydrogel Implant and their In-Vivo Effects on Animal Wound Model
In order to investigate the in-vivo wound healing effects of embodiments of the present invention, two hydrogel implant versions were developed: 7 being blank Genipin-crosslinked PDRN/Sacran hydrogel implants, the other 7 being EGF-loaded Genipin-crosslinked PDRN/Sacran hydrogel implants.
The blank hydrogel implants were constructed as aforementioned, with dimensions (w) 25 mm×(d) 25 mm×(h) 1 mm and a PDRN/Sacran 40/60 content. The EGF-loaded hydrogel implants were also constructed as aforementioned with the addition of loading EGF at 0.001% w/w concentration by dialyzing EGF (Perpetuum) into the PDRN/Sacran solution, following by loading into Genipin Solution, followed by the gelling phase. The EGF-loaded PDRN/Sacran hydrogel implants were constructed with same dimensions, being; (w) 25 mm×(d) 25 mm×(h) 1 mm and a PDRN/Sacran 40/60 content.
The wound healing effects of the present invention were assessed in male adult Wistar rats (Germany, 10 weeks old, 280-320 g, n=21). The rats were housed under a 14 h light/10 h dark cycle with access to water and food “ad libitum”. Before the study, all animals were in quarantine for two weeks. Furthermore, all manipulations were performed under sterile conditions in accordance with German guidelines for the care and use of laboratory animals and under protocol approved by the ethical committee of Charité—Universitätsmedizin (Berlin). Transplantation experiments with blank Genipin-crosslinked PDRN/Sacran hydrogel implants, EGF-loaded Genipin-crosslinked PDRN/Sacran hydrogel implants, and control samples were carried out under anesthesia with isoflurane gas (300-400 mL/min, isoflurane: 1.5-2.0%) using a Univentor anesthesia unit (Univentor).
For the purpose of the study, standard full-thickness wounds were created. Briefly, on day 0, rats were anesthetized, the dorsum were shaved and cleaned using a saline-drenched cotton gauze and further swabbed with 70% isopropyl alcohol. Single full-thickness wounds with dimensions, being; (w) 25 mm×(d) 25 mm×(h) 1 mm, were created on the left dorsal flank skin of each rat to the depth of the loose subcutaneous tissues, and were left open, see
The rats were divided into three groups (7 rats per group). The excised full-thickness wounds of the control group were dressed with sterile, medical gauze. The blank group was treated with the Genipin-crosslinked PDRN/Sacran hydrogel implant, while the EGF-loaded group was treated with the EGF-loaded Genipin-crosslinked PDRN/Sacran hydrogel implant. To prevent the rats from removing the treatment materials, a pief of Sorbaview (Centurion) was placed on top of all wounds. All rats were housed in individual cages, maintained at an ambient temperature (22° C.), with 14 h light/10 h dark cycles and “ad libitum” access to water and food.
For biochemical-, histopathological-, and antioxidant enzyme analysis, 3 rats per group were sacrificed under anesthesia on the 4th and 8th day after surgery. The wound collagen content, granulation tissue formation, wound maturity, and superoxide dismutase and catalase activity were investigated in detail as described hereunder.
For the histopathological alteration evaluation, adjacent skin fragments were removed together with the wound area. Following standard protocol, the collected samples were fixed in 15% buffered formalin (Sigma-Aldrich), embedded in paraffin (Sigma-Aldrich) and then perpendicularly sectioned to the wound surface into thin sections. All tissues were stained with hematoxylin and eosin (Sigma-Aldrich) and analyzed using a light microscope (Thunder, Leica). To visualize macrophages, the tissue sections were also stained with mouse anti-Iba IgG antibodies (Merck), and Alexa488-conjugated anti-mouse IgG antibodies (Thermo Fisher), followed by counter-staining with Hoechst 3342 fluorescent solution (Thermo Fisher), according to the instructions of the manufacturer.
Immediately after wounding of the rats, subsequently after dressing removal, and cleansing with sterile saline solution (Sigma-Aldrich) on the 4th and 8th day after surgery, the wound areas were photographed with a digital camera (Panasonic HC×1000) accompanied by identity plates and calibration bar. Based on scaled digital images of each wound, using imageJ analysis software, the wound closures were determined. By measuring the open wound area in each digital image, at each point, the wound closures were calculated. By using the following formula, the open wound area was calculated as % of the original area immediately after wounding on day 0:
The effects of the blank- and EGF loaded hydrogel implants on the process of granulation and re-epithalization were studied to evaluate wound closure in more detail. In hematoxylin- and eosin-stained tissue samples, the thickness of granulation tissue and the extent of re-epithalization were evaluated. As shown in
Wound healing progression is shown in
By measuring the open wound areas of the rat groups on days 4 and 8, the residual wound surface was determined, see
Obviously, a reduction of the residual wound surface is a key parameter in wound healing, as such it indicates a reduction in inflammation and infection.
To evaluate the effects of both blank PDRN/Sacran and EGF-loaded PDRN/Sacran hydrogel implants on tissue inflammation, each group of rats was tested by Hoechst 33528 and anti-Ibal antibody staining of body tissue samples. Hematoxylin and eosin staining supported the appearance of enhanced wound healing in the groups that were treated with both blank PDRN/Sacran and EGF-loaded PDRN/Sacran hydrogel implants, versus the control group. The inflammatory response at the implantation site was evaluated in order to better understand the healing effect of the implanted materials.
As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 20163463.1 | Mar 2020 | EP | regional |
