Patent Application: US-201113334388-A

Abstract:
a composition of exceptionally dense chitosan and a novel method for producing the dense chitosan structure have been described . the novel production method employs coincident compression and vacuum on a neutralized chitosan polymer that results in an exceptionally dense chitosan film or membrane material . the dense chitosan film or membrane composition possesses multiple physical and clinically appealing qualities for a variety of medical applications on or in animals , mammals , or humans .

Description:
based on the foregoing discoveries , there is provided herein a novel chitosan structure having a density of greater than 0 . 6g / cm 3 , methods of making the composition , and methods of using the composition for the medical uses described in the background of this document . the method of making the chitosan structure can be characterized by the following three sequential steps : b ) neutralizing said solution to form a gel of polymerized chitosan ; in a preferred embodiment the resulting high - density chitosan film or membrane composition has a density greater than 0 . 6 g / cm 3 , and more preferably greater than 0 . 8 g / cm 3 . in a preferred embodiment of the present invention , the chitosan . starting material used in the acidic solution is approximately 70 - 95 % dd . however , the present invention also allows for dd from 56 %- 99 %. in a preferred embodiment the chitosan is present as chitosan base . however , the chitosan may be present as a salt such as chitosan acetate , chitosan succinate , chitosan adipate , chitosan chloride , chitosan glutamate , chitosan lactate , chitosan aspartate , chitosan pyruvate , chitosan phosphate , chitosan glycolate , chitosan ascorbate , chitosan salicylate , chitosan formate , or chitosan malate . in another preferred embodiment of the present invention , the chitosan starting material has an average viscosity of approximately 400 - 500 centipoise ( cps ) or millipascal ( mpas ). however , the present invention contemplates chitosan starting material viscosities from about 5 to 3000 mpas . in a preferred embodiment of the present invention , the chitosan is solubilized in 1 % acetic acid . however , the present invention considers acidic solvents other than acetic acid and solvent percentage ranging from 0 . 1 %- 10 %. for example an appropriate organic acid with ph less than 5 . 0 such as formic , glycolic , citric , or lactic acid would also be suitable . other suitable acids include hydrochloric acid , glutamic acid , aspartic acid , ascorbic acid , pyruvic acid , malic acid , maleic acid , fumaric acid , glucuronic acid , sorbic acid , and folic acid . in a preferred embodiment of the present invention , the chitosan concentration in the solution is 2 - 4 %. however , the present invention contemplates chitosan concentrations of 0 . 1 % to 25 %. in another preferred embodiment of the present invention , the chitosan is solubilized in acidic solvent for 7 days prior to forming a neutralized chitosan . gel ( either with or without a freezing step prior to neutralization ). however , the present invention considers chitosan solutions prepared immediately , or up to 2 years prior to forming the neutralized chitosan gel ( either with or without a freezing step prior to neutralization ). in a preferred embodiment of the present invention , the chitosan solution is poured into a form or mold in an amount at a thickness of approximately 0 . 3 - 0 . 5 g chitosan solution per square cm of the form or mold area . however , the present invention contemplates chitosan solution amounts as low as 0 . 1 g / cm2 or as high as 10 g / cm2 within the mold or form prior to freezing . in a preferred embodiment of the present invention , the chitosan solution is allowed to degas by applying vibration to the solution through the mold or form . vibration time is preferably 10 minutes . however , the present invention contemplates vibration times from 1 second to 10 days . in an alternative embodiment , the present invention contemplates degassing the chitosan solution using an applied vacuum . in a preferred embodiment of the present invention , the chitosan solution is frozen in the mold or form to become a solidified chitosan suspension . in a preferred embodiment of the present invention , the chitosan solution is frozen at approximately − 80 ° c . for 1 h . in an alternative embodiment , the chitosan solution is frozen at approximately − 20 ° c . for 16 h . however , the present invention contemplates freezing of the chitosan solution at temperatures ranging from 0 ° c . to − 276 ° c . for times ranging from 1 minute to 365 days , sufficient to freeze the chitosan solution . the present invention also contemplates the possibility of not freezing the chitosan solution at this stage of the process . in a preferred embodiment of the present invention , the solidified ( if frozen ) chitosan suspension is de - molded ( removed from the mold ) while solid and subsequently immersed in a base , such as 1 - 2m sodium hydroxide , while solid , for 24 h to completely neutralize the acidic solvent within the solidified chitosan suspension , producing a polymerized gel . however , the strength and volume of the base required to completely neutralize the acidic solvent within the solidified chitosan gel , and the duration of the immersion , may vary according to the size and acidity of the solidified chitosan suspension . the present invention contemplates any one of several bases known to those practiced in the science of chemistry , such as sodium hydroxide or potassium hydroxide , having a strength ranging from 0 . 1m to 10m , with an immersion period ranging from 1 minute to 3 months . alternative hydroxides may be used , and they include calcium hydroxide and magnesium hydroxide . in a preferred embodiment of the present invention , the neutralized chitosan gel with a basic ph is washed for 24 h in deionized or distilled h 2 o or aqueous buffer solution to remove the basic solution to become ph neutral or substantially neutral ( e . g . ph 5 - 11 , 5 - 9 or 5 . 5 - 7 . 5 ). however , the present invention contemplates a washing period from 1 minute to 3 months . the present invention also contemplates using continuous flow of deionized or distilled h 2 o or aqueous buffer solution during this rinsing step . the present invention also contemplates not washing the neutralized chitosan gel at all . in a critical aspect of the present invention , the liquid is removed from the neutralized chitosan gel while concurrently compressing the chitosan . dehydration is preferably performed with the use of a vacuum and heat . compression is preferably performed with a minimal linear pressure of 25 inches of hg and is preferably dispersed evenly over the chitosan gel to obtain a uniform membrane . however , compression is contemplated using a minimal linear pressure of 5 - 500 , 10 to 100 , or 20 to 50 inches of hg . dehydration and compression are preferably performed at a temperature of 80 ° c . however , the present invention contemplates dehydrating and compressing the chitosan gel at temperatures ranging from 2 ° c . to 150 ° c ., 40 ° to 120 ° c ., or 50 ° to 100 ° c . of course , it will be understood that dehydration and physical compression can occur in the presence of a vacuum , either by itself or with added heat , in a process known as outgassing . the vacuum applied is preferably less than atmospheric pressure , and as low as 0 . 6 , 0 . 4 or 0 . 2 atmospheres . dehydration and compression are preferably performed for a period of 4 hours . however , the present invention contemplates the performance of dehydration and compression for periods of time ranging from 1 minute to 3 months . in a preferred embodiment of the present invention , the neutralized chitosan gel is placed on or inside a semi - permeable membrane , prior to application of vacuum dehydration . the semi - permeable membrane subsequently facilitates loss of water vapor under vacuum , while preserving the integrity of the dehydrating and dehydrated polymeric chitosan . the semi - permeable membrane is selectively permeable for water , while retaining the molded chitosan gel within the margins or boundaries of the semi - permeable membrane , and may be a cellophane or other cellulosic membrane or another material . the dehydrated high - density chitosan film or membrane may be subsequently removed from the semi - permeable membrane used during the dehydration process . in another preferred embodiment of the present invention , the neutralized and polymerized chitosan gel is immersed in a glycerol solution for a period of time ranging from 1 second to 10 days , then placed on or inside a semi - permeable membrane for vacuum dehydration . furthermore , it is preferred that the glycerol solution contains approximately 5 % to 20 % or 10 % glycerol in water or in aqueous buffer . however , the present invention may use glycerol concentrations ranging from 1 % to 50 % during this process . the resulting chitosan structure preferably takes the form of a film or membrane having a thickness less than 10 mm , 5 mm , 2 mm , 1 mm , or even 0 . 5 mm . the density of the structure , as noted previously , preferably exceeds 0 . 6 g / cm 3 , and may be up to 1 . 6 g / cm 3 . in another preferred embodiment the density of the structure exceeds 0 . 8 g / cm 3 , and may be up to 1 . 6 g / cm 3 . the film or membrane can also be characterized by its ph , which preferably ranges from 5 . 0 to 9 . 5 . the film or membrane can be chopped up or ground and used as particulates , but is preferably used as a film or membrane due to its excellent physical properties ( e . g ., tensile strength , elasticity , and resistance to suture pull - out ). in a preferred embodiment the chitosan film or membrane of this invention does not require a chemical or light - induced cross - linking step , and yet attains a dehydrated density of & gt ; 0 . 6 g / cm 3 and more preferably & gt ; 0 . 8 g / cm 3 . however , for some applications the inclusion of a chemical or light - induced cross - linking step might provide some benefit ( s ), such as reduced biodegradation potential . in another embodiment , the resulting dense chitosan structures of the present invention have physical properties that are beneficial for use in biomedical procedures in an animal , mammal , or human , such as surgically implanted films or membranes . when assessed for tensile strength , elasticity , and / or resistance to suture pull - out , the dense chitosan materials demonstrate excellent physical characteristics . astm international standard methods have been established for the evaluation of these physical parameters for thin films or membranes ( astm 2002 ; astm 2006 ). these standard methodologies with minor modifications ( e . g ., for tensile testing strips semi - circular rather than semi - oval template cut - outs according to astm standard method . d 1708 - 06a , and having a minimum width of ˜ 2 . 5 mm ( astm 2006 ); for suture pull - out strips having a width of ˜ 5 mm ) have been utilized to characterize the resulting dense chitosan structures of the present invention . the present invention discloses a composition comprising chitosan in a film or membrane having a density greater than 0 . 6 g / cm 3 , and more preferably greater than 0 . 8 g / cm 3 . in another preferred embodiment the chitosan composition has a ph of from 5 . 0 to 9 . 5 . in another preferred embodiment the chitosan composition includes glycerol . finally , the invention provides methods of treatment using the structures of the present invention , and can thus be defined as a method of treatment comprising : providing a chitosan composition having a density greater than 0 . 6 g / cm 3 ; and placing said composition on or within an animal . in preferred embodiments the animal is a mammal or human , and in another preferred embodiment the structure is hydrated in water or a buffered aqueous solution and in the presence or absence of one or more compounds selected from a pharmaceutical , a biologic agent , a nucleic acid , a vaccine , an immune effector , or a salt thereof prior to use . in another preferred embodiment the chitosan composition serves as a physical barrier film or membrane to separate tissue layers within an animal . in another preferred embodiment the film or membrane on or within the animal , mammal , or human resorbs over time , and the rate thereof is in part dependent upon the dd and thickness of the material . in another preferred embodiment the chitosan composition serves as an anti - infective physical barrier film or membrane on or within an animal . in another preferred embodiment of the present invention , the chitosan film or membrane is permeable to small molecules in water or aqueous solution . in another preferred embodiment of the present invention , the physical properties ( e . g ., tensile strength , elasticity , and resistance to suture pull - out ) alone or in addition to clinical handling characteristics ( e . g ., wet - ability , conformability to surgical implant sites , and suture - ability ) facilitate excellent ease - of - use of the resulting dense chitosan films or membranes in clinical settings in an animal , mammal , or human . in the preferred method of freezing the chitosan solution prior to neutralization , ultra - freezing a chitosan solution of approximately 0 . 3 - 0 . 5 g / cm 2 in the mold at − 80 ° c . for an hour ultimately results in polymerization of the chitosan . on the exposed top surface with a woven , fibrillar , porous , structure at that surface when examined by microscopy or scanning electron microscopy . the resulting dehydrated film or membrane has an overall density & gt ; 0 . 6 g / cm 3 and more preferably & gt ; 0 . 8 g / cm 3 and is somewhat asymmetric , with a smoother , less fibrillar surface on the alternate , bottom side . in the preferred method of freezing the chitosan solution prior to neutralization , ultra - freezing at − 80 ° c . for significantly more than an hour ( e . g ., two hours ) can result in physical cracking of the frozen chitosan gel and of the final membrane structure . in the preferred method of freezing , the freezing of the chitosan solution within the mold at − 20 ° c . prior to neutralization , reduces the extent of woven structure of the final membrane structure . regardless of freezing temperature , the resulting membrane has a density & gt ; 0 . 6 g / cm 3 . in the absence of freezing the chitosan solution prior to neutralization , the resulting compressed and dehydrated membrane has no visible woven , fibrillar structure . regardless of freezing or lack thereof , the resulting membrane has a density & gt ; 0 . 6 g / cm 3 . the relevance of freezing the acidic chitosan solution prior to neutralization of the acid and dehydration with compression is further exemplified by mechanical properties of the materials generated with and without the freezing process . in measuring the resistance to suture pull - out using an instron machine , chitosan membranes prepared without the freezing process had an inferior pull - out force of 2 . 0 n ± 0 . 3 n / mm of membrane thickness , while membranes of the same composition prepared with a freezing temperature of − 80 ° c . for 1 h prior to neutralization had a superior resistance to suture pull - out of 4 . 5 n ± 0 . 1 n / mm of membrane thickness . exemplifying the importance of neutralizing the frozen chitosan suspension with alkali to a semi - solid gel prior to dehydration and compression , attempts at evaporating while compressing acidic chitosan solutions have failed . dehydration with compression requires a semipermeable membrane ( e . g ., cellophane ) to retain the solute ( chitosan polymers ) while allowing passage of the solvent with a gradual increase in the density of the chitosan while drying . a plausible reason for the inability to dehydrate an acidic chitosan solution through a semi - permeable membrane is that the viscous unpolymerized chitosan eventually accumulates at the surface of the membrane and blocks the passage of the solvent . the result is failure to dehydrate the solution , even in the presence of heat . for the same reasons , freezing of the acidic chitosan solution immediately followed by dehydration and compression also fails . for the same reasons , dehydration with compression of a wet acidic chitosan sponge ( produced by lyophilizing the acidic chitosan suspension ) also fails , failing to dry with vacuum dehydration . for the same reasons , dehydration with compression of a wet , acidic air - dried chitosan structure also fails . polymerization of the chitosan suspension by neutralization in alkalai prior to vacuum compression is essential . exemplifying the importance of evaporating and compressing a neutralized chitosan gel , attempts at compressing a dry lyophilized acidic sponge resulted in a cracked chitosan membrane structure . wetting the dry compressed membrane results in unacceptable recoil swelling and loss of the unpolymerized membrane structure . the importance of maintaining a ph greater than 5 . 0 following polymerization of the chitosan solution in a strong base into a gel is exemplified by the loss of chitosan gel structure when the neutralized chitosan gel is placed in an acidic solution ph 2 . 9 for 20 h resulting in disintegration of the chitosan gel structure . the importance of maintaining a ph environment greater than ph 5 . 0 following dehydration of the neutralized chitosan gel is exemplified by the complete loss of dense chitosan film or membrane structure following 24 h in an acidic environment of ph 4 or less . exemplifying the importance of dehydrating and compressing a neutralized chitosan gel and not a lyophilized sponge , compression of a wetted neutralized lyophilized chitosan sponge results in insufficient chitosan density of 0 . 38 g / cm 3 . compression of a dry neutralized lyophilized chitosan sponge results in insufficient chitosan density ( 0 . 065 g / cm 3 ). exemplifying the importance of dehydration with compression , concomitantly , experiments where compression was provided in the absence of adequate dehydration during the compression resulted in a fissured and unsatisfactory chitosan final structure . the effect of vibration on the acidic chitosan solution prior to neutralizing has no effect on final membrane structure . exemplifying the biological relevance of chitosan films or membranes with high density , these membranes demonstrated permeability to small molecules . for instance , high density chitosan films or membranes prepared from 4 % chitosan solution were permeable to methylene blue and crystal violet ( mw 285 and 373 , respectively ) in phosphate buffered saline ( pbs ) solution using franz cell technology . this demonstrates that the films or membranes are permeable to selected small molecules . permeability to nutrients on or within an animal , mammal , or human may have physiologic benefits . further exemplifying the biological relevance of chitosan films or membranes with high density , living mammalian cells were seeded and maintained on the membranes for at least 3 days in culture . cell binding and cell compatibility were observed on both sides of the membrane . evidence of proliferation and cell migration were also demonstrated . keratinocyte migration was most evident on the smoother surface bottom side while in the mold ) relative to the more porous surface ( i . e ., top side while in the mold ) as measured by microscopic image analysis . these results provide evidence of in vitro biocompatibility . further exemplifying the biological relevance of chitosan films or membranes with high density , wound healing in a mammalian model was observed when the membranes were surgically placed at the base of full - thickness surgically - induced ulcers of the oral palate in rats . healing was associated with re - development of collagen in the subepithelial matrix and re - epithelialization of the ulcers . histological analysis after membrane implantation for 1 to 12 weeks also indicated that the high - density chitosan is biocompatible , while biodegradable or resorbable . establishment of a physical barrier on or within an animal , mammal , or human that is resorbed over time has clinical utility . for instance a physical barrier between dissimilar tissues ( e . g ., bone vs . soft tissue ) can facilitate differential rates of healing on the opposite sides of the film or membrane . furthermore , in view of in vitro enzymatic degradation rates , the rates of resorption in vivo ( see below ) are similarly anticipated to be dependent upon the percentage of dd and / or thickness of dense chitosan films or membranes . in other words , the rates of degradation can be “ controlled ” at least in part by varying the percentage of dd and / or thickness . exemplifying the relevance of density to clinical functionality and utility is the strong correlation between densities of dry chitosan films or membranes and other physical properties , such as tensile strength . the astm standard methods with minor modifications ( e . g ., for tensile testing strips semi - circular rather than semi - oval template cut - outs according to astm standard method d 1708 - 06a , and having a minimum width of ˜ 2 . 5 mm ( astm 2006 ); for suture pull - out strips having a width of ˜ 5 mm ) have been utilized to characterize the resulting dense chitosan films or membranes . the dense chitosan films or membranes produced by the methods claimed herein show a direct correlation between membrane density and tensile strength . in general the dense chitosan films or membranes of the present invention when tested with an array of experimental variables from production batch to batch ( e . g ., amount of starting chitosan solution , dry membrane thicknesses of less than 1 mm and typically 0 . 2 to 0 . 6 mm , percentage of dd from 70 % to 95 %, source materials from different vendors , post - drying process modifications if any , etc .) yield in tests using an instron machine the following typical ranges of physical properties : ( a ) maximum tensile load of approximately 2 to 14 n (˜ 2 . 5 mm minimum width ); ( b ) maximum tensile stress of approximately 20 to 140 mpa (˜ 2 . 5 mm minimum width ); and ( c ) suture pull - out maximum load of approximately 0 . 5 to 4 . 5 n (˜ 5 mm width ). furthermore exemplifying the relevance of physical characteristics of dense chitosan films or membranes of the present invention to clinical utility is the combination of density , tensile strength , elasticity , and resistance to suture pull - out , some or all of which are desirable features for suture - able implantable surgical membranes . exemplifying the relevance of chitosan deactylation pertaining to the methods disclosed and claimed herein , degradation in a concentrated lysozyme solution buffered at ph 6 . 5 and 37 ° c ., was complete within 8 days for a 70 % dd membrane , complete within 11 days for a 75 % dd membrane , partially complete within 18 days for 80 % and 85 % dd membranes , and not evident in 90 % and 95 % membranes after 3 weeks under these conditions . these results indicate that the inherent susceptibilities of the starting polymeric materials ( i . e . chitosan powder of varying percentage of dd ) to enzymatic degradation in vitro have not been destroyed by the processes of the present invention while producing dense chitosan films or membranes . furthermore , dense chitosan films or membranes of the present invention remain labile to acid depolymerization ( and solubilization ) without enzymatic degradation when placed in acetic acid solution or a buffered solution at or below ph 4 . exemplifying the relevance of treating the neutralized and polymerized chitosan gel with a glycerol solution ( e . g ., 10 or 50 percent glycerol in water ) prior to the dehydration step , the resulting film or membrane has high density similar to film or membrane produced without this glycerol solution step , and with beneficial high tensile strength , resistance to suture pull - out , and handling characteristics , for instance flexibility and ease of cutting . this combination of attributes ( i . e ., physical properties and clinical handling characteristics ) provide a film or membrane material of great utility for use on or in an animal , mammal , or human . throughout this application , various publications are referenced . the disclosures of these publications are hereby incorporated by reference in order to more fully describe the state of the art to which this invention pertains . it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention . other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein . it is intended that the specification and examples be considered as exemplary only , with a true scope and spirit of the invention being indicated by the following claims . abou - 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