Patent Application: US-9491206-A

Abstract:
the invention relates to a novel method for production of nanofibrillar chitin , sustainable from an industrial manufacturing standpoint and suitable for producing chitin nanofibrils having improved properties and free from less crystalline components . the invention also relates to novel chitin nanofibrils obtained with said method and characterized by an increased dispersibility in aqueous media . the invention further relates to uses of nanofibrils in pastes and aqueous gels useful for topical on - skin application , to the manufacturing of materials of biomedical interest , as well as to the deposition of chitin nanofibrils on coating surfaces or the incorporation of the same inside of items like , e . g ., fibers and fabrics of any origin , natural as well as synthetic or mixed ones .

Description:
the starting product for the compounds of the invention is crustacean chitin , for instance crab alpha - chitin , commercially available in the form of powder or flakes . other types of chitin may be used as well . pilot - scale production of spray - dried chitin nanofibrils opens new perspectives to applications , considering that known applications to date are purely theoretical owing to product scarcity and to the incomplete definition of its grade . the present invention not only describes a novel method for production , but also novel chitin nanofibrils that , having been obtained by spray - drying , are susceptible of being brought into suspension in aqueous and organic environments and stably kept over long times . these features , i . e . the high dispersibility and stability of the suspensions obtained , paves the way to numberless practical applications . in a first step of the process , chitin powder is boiled under reflux with stirring in a strong acid , preferably 3 m hcl , for a sufficient time , usually of 90 min : after a brief rest , chitin settles spontaneously and it is easy to remove nearly all of the supernatant acid with a pump . in a second step , it is added a water volume equal to that of the acid removed , and the resulting suspension is dialyzed against water for a 20 - hour time , or treated by hollow fiber ultrafiltration according to techniques known to those skilled in the art . then , the suspension is centrifuged at a low number of rpm ( about 3000 ) with a standard centrifuge to remove non - hydrolyzed chitin particles , whereas nanofibrils remain in suspension . the centrifugation yields : a solid chitin powder residue , intended to be subjected to further treatment as in the first step , and the colloidal suspension of chitin nanofibrils intended for the subsequent spray - drying treatment . the above - mentioned solid chitin powder residue , discarded by the preceding authors , proved particularly sensitive to a second treatment with fresh 3 m hcl , in which it is nearly totally transformed into nanofibrils . the colloidal suspension of nanofibrils , preferably obtained by reuniting the first suspension with that produced by recycling the solid residue , is pumped through the sprayer at about 150 ° c . for a few seconds , and immediately cooled to about 40 ° c . dry product yield is about 60 % of the theoretical one . high inlet temperature ( about 150 ° c .) notwithstanding , it has surprisingly been found that the nanofibrils thus produced undergo minimal alteration of their water sphere and of the crystallization water , as exposure to that temperature is very short . in fact , the spray - dried nanofibrils are characterized by a crystallization water content ranging from 5 to 10 % and a hydration state ranging from 10 to 15 %. thanks to these characteristics , the nanofibrils of the invention , though rolled up into structures resembling microspheres , are not aggregated and spontaneously redisperse in water even after months of storage in a closed container . thermogravimetric curves recorded on such samples demonstrate a different and more favourable hydration state compared to known lyophilized or hot - dried nanofibrils described in the prior art . analytical and control methods required for implementing the invention and identifying and characterizing the nanofibrillar chitin are : infrared ( ir ) spectrophotometry , x - diffraction spectrometry , thermogravimetry and electron microscopy . the ir spectra of chitin nanofibrils contain all of the bands characteristic of chitin with the due intensity , in particular those at 1556 , 1622 , 1658 , 2891 , 3107 , 3262 and 3439 cm − 1 , as shown by nair & amp ; dufresne ( 2003 ), lu et al . ( 2004 ) and persson et al . ( 1992 ). however , the chitin nanofibrils produced by the present method are more crystalline than those produced to date for analytical purposes . in fact , by comparing the spectrum in fig1 with the spectra published by the cited authors ( fig3 , 4 and 5 ), it is evident that in former several bands are present that are indistinguishable in the spectra of the preceding authors , exactly those at 1204 , 975 , 917 cm − 1 . moreover , the bands at 1156 , 1116 , 1075 and 1029 cm − 1 are well - resolved , whereas in the preceding spectra they are hazy . even the two most important bands qualifying the chitin as alpha , i . e . the bands at 1656 and at 1622 cm − 1 , are much more definite and resolved . lastly , the signals in the 3855 - 3652 cm − 1 range indicate an improved hydration state . the dimensions of the nanofibrils are assessed through transmission electron microscopy ( tem ) technique , by means of an image analyzer and the drafting of a histogram showing length distribution : the 250 - nm length is present in 25 % of the nanofibrils ; the 200 - nm one in 23 % thereof , the 300 - nm one in 18 % thereof , etc ., with maximum values of 450 nm for 3 % thereof . the measuring of the degree of crystallinity of the nanofibrils obtained is carried out through x - ray diffraction spectrometry technique , and this is correlated to the area subtended by the diffraction peaks . the values of the nanofibrils of the invention , reported in fig2 , are summarized in the following table : in particular , the spectrum in fig2 indicates that the nanofibrils produced by the present method are highly crystalline , as peaks are present that in normal chitin samples are depressed or scarcely evident , in particular those at 0 . 7044 and 0 . 3441 nm . the other two major peaks are of strong intensity and well - defined . lastly , thermogravimetry by suitable programmed - temperature balance allows to detect in derivative thermograms first the water released in the 70 - 180 ° c . range , and the reading of the temperature ( near to 300 ° c .) at which pyrolysis starts . the spray - dried nanofibrils of the invention instantaneously yield highly homogeneous dispersions . surprisingly , this characteristic was detected , for the first time , also in connection with suspensions of nanofibrils in viscous aqueous solutions , of which the nanofibrils can enhance the biological properties without increasing the viscosity — in fact , an excessive increase of the latter would prejudice their use . as the chitin nanofibrils of the invention exhibit an enormous surface development , allowing them to interact with enzymes , platelets and other compounds or cells present in living tissues , addition of nanofibrils of the invention to viscous solutions accordingly increases the content of biochemically active material without restricting its practical applicability . the spray - dried chitin according to the invention keeps all general properties of chitin . it is highly biocompatible with human tissues , is not recognized as an extraneous body ; is biodegradable , as already highlighted by works with films of chitin implanted in rat &# 39 ; s back ; or as suture thread ; is slowly reabsorbable also in human tissues by means of lysozime ; influences collagen &# 39 ; s proline - hydroxyproline ratio ; fosters proliferation of fibroblasts , collagen producers ; is not antigenic , instead fostering the organism &# 39 ; s immunologic response ; is a haemostatic agent ; following intravenous administration as a thin - particle suspension causes fagocytosis by macrophages . moreover , the suspensions of spray - dried chitin form , after removal of dispersion liquids , resistant , reabsorbable and tissue - compatible films . thanks to these numberless properties , the spray - dried chitin nanofibrils have medical - surgical , generally clinical and cosmetic - dermatological applications . in particular , they find application in a protective or curative treatment of skin abrasions , wounds , burns , particularly in a treatment for supporting and stimulating hemostasis , tissue healing and regeneration processes of injured tissues . moreover , the compositions of the invention find application in the cosmetic surgery field , in particular in the dermocosmesis field , as cutaneous fillers for the treatment of wrinkles and other cutaneous irregularities and blemishes through subcutaneous hypodermic injection , or in the cutaneous treatment through masks , films , sponges . in the use as cutaneous filler , the compositions of the invention , with the due technical skills , can be used in association with other natural and synthetic biocompatible polymers , like , e . g ., hyaluronic acid , polylactic ( pla ) or polyglycolic ( pga ) acid with copolymers thereof ( plga ) or with other biomimetic hydrogels such as polyethylene glycols ( pegs ) or oligo - polyethylene glycol fumarate ( opf ), etc . suitable pharmaceutical or cosmetic compositions comprise the spray - dried nanofibrils in a dry state , or liquid , fluid or viscous suspensions , gel , paste , or any other form allowing a practical topical application on skin or mucosae . the suspensions of nanofibrils may contain thickeners or plasticizers , like glycerol , sorbitol , mannitol or other polyols , chitosan , chitosan glycolate , hydroxymethylglycinate , hydroxyethyl cellulose or dibutyryl chitin . a practical embodiment of the present invention is a fluid or viscous gel of chitosan to be used as liquid dressing or tissue - healing antibacterial and antifungal even in the form of spray , in which there are suspended chitin nanofibrils that , without increasing the viscosity , greatly increase the chitin / chitosan content in terms of weight percent . the suspensions of nanofibrils of the invention are placed into containers equipped with devices or means for both an intra / subcutaneous application , e . g . hypodermic syringes , and a topical one on - skin or on mucosae : e . g ., spray cans , nebulizers , pencils , brushes or other usual means capable of depositing on - skin a liquid thin layer , which , after drying , generates a film of material , protective and absorbable over time , to protect wounds , abrasions , irritations or burns . the spray form is particularly suitable for the treatment of large - sized wounds or burns . moreover , the fact that chitin nanofibrils possess weak cationic character , enables to adsorb them on spongy surfaces or on films having anionic character , e . g . modified cellulose like carboxymethyl cellulose , or polyacrylate , or on natural yarns like wool , cotton , flax , hemp , jute , or synthetic yarns . suchlike articles treated with suspensions of nanofibrils are susceptible of dermocosmetic use by means of face masks or fabrics in general , which , into contact with skin , prevents forms of allergy and / or allergic sensibilization . these articles differ from those described in the prior art , as the former do not incorporate chitin nanofibrils thereinside , rather having the surface covered with nanofibrils . optionally , the compositions of the invention may also contain bactericides , fungicides , antimicrobials like , e . g ., chlorhexidine hydrochlorate , triclosan , or metallic silver or other antimicrobials of different nature , alpha - amino acids , beta - amino acids or derivatives thereof like taurine , carotenoids , such as lutein , moisturizers , such as glycine and other pharmacologically active principles . suspensions of spray - dried chitin nanofibrils containing these substances can generate , by evaporation of the aqueous phase , mechanically resistant films used as cosmetic beauty masks or for various medical uses . in - film incorporation of spray - dried chitin nanofibrils and synthetic , semisynthetic and natural polymer fibers . a further application of the spray - dried chitin nanofibrils relates to the manufacturing of films , fibers and other solid items . incorporation of nanofibrils is carried out through known techniques , i . e . spinning ( melt - spinning , dry - spinning or wet - spinning ), extrusion ( coextrusion , film - making from solutions , calendering ) and hot - forming . prior art chitin in the form of granules , though fine - sized , is scarcely compatible with generally hydrophobic synthetic polymers , and tends to agglomerate in the extruder , forming globules . on the contrary , the spray - dried chitin nanofibrils suitably mixed to the mass being heated and melt , arrange themselves more evenly , generating a homogeneous and stable dispersion . contents of spray - dried chitin nanofibrils , up to 70 % by weight , are added to polymers like : polyhydroxybutyrate , polyhydroxybutyrate - covalerate , polylactic , polyglycolic , poly - epsilon - caprolactone and copolymers thereof , and other polyesters like polybutylene succinate , polybutylene terephtalate adipate , during the melting ( up to 300 ° c .) in a co - rotating twin - screw extruder ( brabender , or the like ), in the absence or in the presence of plasticizers . thus , there are obtained products in the form of film having features of improved applicability in the biomedical and the packaging field . spray - dried chitin nanofibrils may easily be added to any biodegradable polymer for medical use , and also to semisynthetic polymers like the polylactic and polyglycolic acid , or hydroxyethyl cellulose or dibutyryl chitin . the latter easily forms transparent , mechanically resistant and biocompatible films , it being soluble in various organic solvents like methanol , ethanol , acetone , chloroform , etc . the presence of dibutyryl chitin in ethanol prevents any disturbance to the spray - dried chitin nanofibrils , so much so that they easily disperse in the dibutyryl chitin solutions . solvent evaporation yields white films firmly adhering to many materials , like steel , plastics of various nature , glass , wood , paper , etc . the spray - dried chitin nanofibrils may easily be added also to aqueous solutions of natural polymers , preferably cationic and neutral ones , such as chitosan or chitosan glycolate , or to any biodegradable polymer for medical use and also to semisynthetic polymers like hydroxyethyl cellulose or dibutyryl chitin , or polylactic and polyglycolic acids , the latter two when already polymerized or under polymerization . dibutyryl chitin , by being soluble in several organic solvents like methanol , ethanol , easily forms transparent , mechanically resistant and biocompatible films . lastly , the nanofibrils of the invention may be adsorbed on natural yarns , like wool , cotton , flax , silk ( tel quel and regenerated ), hemp , jute , for preparing fabrics with antiallergic characteristics typical of chitin . absorption on yarn surface occurs either through immersion in colloidal suspensions of nanofibrillar chitin or by spray treatment of the yarns or the fabrics themselves . presence of minimum amounts of metallic silver is known to afford antimicrobial activity to various materials . it has surprisingly been found that the silver ion attaches onto the chitin nanofibrils , remaining thereon even after chemical reduction . in example 6 there are adopted conditions and silver concentrations high enough to display its presence by means of a deep black colour , however in practical applications it is not necessary to apply enough silver to blacken the sample ; in fact , antimicrobial properties associable to silver are such as to give merely a pearly hue . chitin nanofibrils adsorb silver ion , which collapses their suspension . this is not obvious , so much so that chromium trichloride does not precipitate the suspensions ( violet colour ), whereas copper sulfate aggregates the nanofibrils without having them colouring appreciably . applications of this aspect comprise the chelation of metals , in particular transition ones , for the scopes that are evident to those skilled in the art , such as the recovery of precious metal traces , the elimination of metal traces from polluted environments , the preparation of materials for electronics and nanotechnologies , etc ., it being evident that the performances of chitin in the nanofibrillar form are exceptionally better than those described on this subject for commercial raw chitin . some inorganic salts and some organic compounds have the property of aggregating spray - dried chitin nanofibrils , as indicated above , whereas other compounds do not influence the dispersion state of spray - dried chitin nanofibrils . a significant case is that of aqueous solutions of alpha - amino acids , ubiquitous in living systems , or of beta - amino acids or derivatives thereof , like taurine , or of organic solutions of carotenoids , like lutein , them also present in animals and vegetables . suspensions of spray - dried chitin nanofibrils containing the above - indicated substances can generate , by aqueous phase evaporation , mechanically resistant films . these films may be added , e . g ., with glycine , thereby performing an intense rehydrating activity if used as cosmetic beauty masks , or be pre - treated with bactericides and fungicides , generating films useful for several medical uses . 10 g katakura chitin are placed in a flask containing 0 . 3 m hcl ( 300 - 800 ml ). content of the condensing section - equipped flask is boiled for 90 min . during cooling , chitin settles on flask bottom ; this facilitates the removal , by means of a pump , of hcl in excess ( about 200 - 600 ml , containing no chitin after dialysis ). from the flask , the chitin and the scarce hydrochloric supernatant are poured into a glass , reconstituting their initial volume of 300 - 800 ml with water . this suspension is dialyzed for 20 hours against water : three changes of water , initial weight : 845 g ; final weight : 873 g ; final ph of 3 . 3 , become 4 . 2 after water addition . this suspension is centrifuged at 3000 rpm to sediment large - sized chitin particles : the supernatant containing the nanofibrils in suspension is sent to the spray - dryer by means of a peristaltic pump . the chitin particles are instead recovered with fresh 3 m hydrochloric acid and treated again . operative conditions of buchi mini - 90 spray - dryer : compressed air supply : 500 ; heating : 9 ; inlet temperature : 148 ° c . ; outlet temperature : 87 ° c . ; peristaltic pump flow rate : 400 ml / hour ( for these conditions flow cannot be greater ). cover temperature : 42 ° c . ; collection vessel temperature : 36 ° c . formulation of nanofibrils with chitosan glycolate and sodium hydroxymethylglicinate , and / or sodium glycinate sodium hydroxymethylglycinate ( 50 %) is an antimicrobial useful for cosmetic formulations , produced by sutton , nj , usa ( cas no 70161 - 44 - 3 ). 0 . 3 m hcl ( 5 . 5 ml ) is added to 50 % sodium hydroxymethylglicinate ( 10 ml ) to adjust its ph , which from 11 becomes 7 ( neutral hydroxymethylglicinate ). chitosan powder ( 3 g ) is wetted in 45 ml water , and glycolic acid ( 1368 mg ) is added . this solution is divided into 6 aliquots of about 7 . 5 g ( each one containing 0 . 5 g chitosan , 7 . 5 ml water and 228 mg glycolic acid ). 1 ) to the first aliquot of chitosan glycolate it is added neutral hydroxymethylglycinate ( 85 microliters ) prediluted in water ( 2 . 5 ml ); final ph of 4 . 5 . 2 ) to the second aliquot it is added neutral hydroxymethylglycinate ( 170 microliters ) prediluted in water ( 2 . 5 ml ); final ph of 4 . 8 . 3 ) to the third aliquot it is added neutral sodium glycinate ( 170 microliters ) presolubilized in water ( 2 . 5 ml ); final ph of 4 . 8 4 ) to the fourth aliquot it is added neutral hydroxymethylglycinate ( 255 microliters ) prediluted in water ( 2 . 5 ml ); final ph of 5 . 0 . 5 ) to the fifth aliquot there are added nanofibrils ( 100 mg ) suspended in water ( 2 . 5 ml ), then neutral hydroxymethylglycinate ( 85 microliters ); final ph of 4 . 5 . 6 ) to the sixth aliquot there are added nanofibrils ( 100 mg ) suspended in water ( 2 . 5 ml ), then neutral sodium glycinate ( 85 microliters ); final ph of 4 . 5 . it can be observed that aliquots 1 , 5 and 6 gelled rapidly and achieved the same consistency ; no difficulties were experienced in dispersing the nanofibrils in the presence of chitosan glycolate , and at about 26 ° c . no undesirable effects such as syneresis or aggregation were observed at + 3 months . aliquots 2 , 4 and 6 achieved higher consistency with respect to 1 , 3 and 5 . however , 1 and 5 contained hydroxymethylglycinate in a concentration recommended for antimicrobial activity , whereas the others contained an excess thereof . aliquots 3 and 6 , produced from sodium glycinate , carry out a low antimicrobial activity . on the contrary , they carry out an intense bactericidal - fungistatic activity if added with 0 . 4 % chlorhexidine hydrochlorate . moreover , aliquot 3 is the most suitable for the manufacturing of a spray product , above all if added also with microbicides , like , e . g ., chlorhexidine . in short , it was observed that neutral hydroxymethylglicinate has the ability to gel chitosan glycolate solutions , presumably thanks to new hydrogen bonds between the components , with no local precipitation of the chitosan ( not even transitorily ). according to the producer , neutral or slightly acid hydroxymethylglicinate has the same antimicrobial abilities of the original one at a ph of 11 . the gel under 5 ) is also advisable for a ready administration of chitosan and chitin to wounds , even bleeding ones , to induce hemostasis and regeneration of injured tissues , as is also that at 3 ) if used as a spray product . aliquots 3 and 6 ( as spray and as gel , respectively ) added with 0 . 4 % chlorhexidine hydrochlorate are active both on gram + and gram − bacteria and on fungi , besides carrying out an intense tissue healing and repairing activity . a clear colourless solution is prepared by mere mixing of dibutyryl chitin ( 36 mg ) into ethanol ( 2 g ). this solution is evaporated in a glass petri dish , yielding a transparent and mechanically resistant film of 2 . 5 cm in diameter . analogous solution is added with spray - dried chitin nanofibrils ( 20 mg ), immediately yielding a suspension that does not settle within the time interval required for preparing the film : this suspension is evaporated in a glass petri dish , yielding a white and mechanically resistant film of 2 . 5 cm in diameter . the same operation attempted with lyophilized chitin nanofibrils does not succeed , as it is impossible to obtain a suspension . semisynthetic polymers with chitin nanofibrils : hydroxyethyl cellulose and other types of thickeners hydroxyethyl cellulose is available in several presentations : for this application it was selected the one marketed as tylose h - 10000 - p2 , produced by clariant gmbh , widely used in cosmetic formulations . chitin nanofibrils ( 2 . 5 g ) obtained as above were placed in water ( 40 ml ); it was added said hydroxyethyl cellulose ( 2 . 0 g ), bringing the temperature to 40 ° c . for 20 min , under gentle stirring . a consistent white gel was obtained , stable even at + 5 months , i . e ., without neither syneresis nor microbial growth , easily spreadable , with fair mucoadhesiveness , which rapidly dries on - skin , becoming transparent . crustacean chitosan powder ( 0 . 5 g ) is suspended in water ( 7 . 5 ml ), and to the suspension it is added glycolic acid ( 0 . 23 mg ), obtaining a clear solution . a suspension of spray - dried chitin nanofibrils ( 102 mg ) in water ( 2 . 5 ml ) is added thereto , obtaining a viscous homogeneous suspension , from which incorporated air is removed by negative pressure . this suspension is placed , on a petri dish , in a stove at 40 ° c . to evaporate the water , obtaining a chitosan film incorporating spray - dried chitin nanofibrils . chitin nanofibrils ( 151 mg ) were added to aqueous silver acetate ( 11 mg in 25 ml water ): at + 3 hours , sediment was ultracentrifuged , abundantly washed with water ( step repeated twice ). the following assays were performed with sodium boron hydride , capable of reducing silver ion to zerovalent state : ( a ) supernatant , pale yellow colour ; ( b ) silver acetate solution , black colour ; ( c ) nanofibrils after ultracentrifugations and washings , dark green colour ; ( d ) control solution , containing silver acetate and nanofibrils , black colour . the silver ion , once reduced to metallic silver , remains adsorbed on the nanofibrils . a clear colourless solution of taurine ( sulphurised beta - amino acid ) ( 100 mg ) in water ( 5 ml ) is prepared , to which it is added a suspension of spray - dried chitin nanofibrils ( 100 mg ) in water ( 2 . 5 ml ), obtaining an uniform suspension that does not settle . a clear , orange / red dyed ( carotenoid , xanthophyll dye and antioxidant ) lutein ( 100 mg ) solution in ethanol ( 5 ml ) is prepared , to which spray - dried chitin nanofibrils ( 100 mg ) are added , obtaining an uniform suspension that does not settle . likewise when lutein dissolved in oils like thymol and carvacrol is used . 1 ) belamie e , davidson p , giraud - guille m m . structure and chirality of the nematic phase in alpha - chitin suspensions . journal of physical chemistry . b , 108 , 14991 - 15000 , 2004 . 2 ) giraud - guille m m , belamie e , mosser g . organic and mineral networks in carapaces , bones and biomimetic materials . comptes rendus palevol , 3 , 503 - 513 , 2004 . 3 ) jollès p , muzzarelli r a a . chitin and chitinses . birkhauser , basel , 1999 . 4 ) li j , revol j f , naranjo e , marchessault r h . effect of electrostatic interaction on phase separation behavior of chitin crystallite suspensions . international journal of biological macromolecules , 18 , 177 - 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