CN104144692A

CN104144692A - Composition, preparation, and use of dense chitosan membrane materials

Info

Publication number: CN104144692A
Application number: CN201280069003.XA
Authority: CN
Inventors: A·A·德卡罗; A·埃利斯; T·P·杜利; M·贝罗索瓦
Original assignee: A Jinta Biotech Co
Current assignee: A Jinta Biotech Co; Agenta Biotechnologies Inc
Priority date: 2011-12-22
Filing date: 2012-12-19
Publication date: 2014-11-12
Also published as: EP2793908A1; KR20140122231A; IL233290A0; JP2015511214A; WO2013096448A1; BR112014015444A2; EP2793908A4

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

Compositions, preparation and the purposes of fine and close chitosan film material

Open field

The present invention relates to fine and close chitosan film or membrane material and preparation method thereof.These materials can be used for medical industries, science and technology industry and other industrial in.

Background

Functional living being investigation of materials has concentrated on exploitation for the improved support of wound healing and organizational project.Many biodegradable polymer are studied for wound healing and organizational project application as support, and comprise synthetic polymer, as polycaprolactone, poly-(lactic acid-altogether-glycolic), PEG; And natural polymer, as alginate, gelatin, collagen protein, starch and chitosan.Wherein, the polymer of natural origin merits attention especially, because as the natural constituents of biological structure, it is similar to natural tissues on biological and chemical.In this situation, have been found that chitosan can be used as the noticeable material standed in many application, and except antibacterium, antifungal and haemostatic properties, also there is unique biological nature, comprise biocompatibility, for the biological degradability of harmful sugared product, nontoxic, physiology's inertia, for the remarkable affinity of protein.

Have the chitosan application of record should date back to for 19th century, Rouget is when discussing the deacetylation form of chitosan in 1859.Chitin (raw material of chitosan) is one of the abundantest organic material of content, and the amount producing by biosynthesis is every year only second to cellulose.It is animal, especially ectoskeletal a kind of important component of crustacean, Mollusca and insecticide.Main fibril polymer in it or some fungal cell wall, and can be produced by microalgae.The chitin derivative of deacetylation is called as " chitosan ".When these two terms are used in 19th century (1800 ' s) first; it is believed that chitin and chitosan are in fact usually to exist as unique and unconverted chemical substance completely different, that fully determine, wherein chitin be complete acetyl group and chitosan be the compositions of complete deacetylation.Yet, after approximately oneth century, find that term " chitin " and " chitosan " is in fact indefinite.In fact these terms refer to the family of the compound that represents visibly different physics and chemistry characteristic, but not refer to fully definite compound.These differences are owing to different molecular weight and the different acetyl group degree of these products.

Chitosan is linear polysaccharide, and glycosamine and N-acetyl glucosamine cell formation by connecting by β (1-4) glycosidic bond, be the linear formula of sugar unit substantially.Depend on source and preparation procedure, its molecular weight generally arrives and surpasses within the scope of 1000kDa at 10kDa.The molecular weight of chitosan polymer is normally determined by viscosity and be take centipoise (Centipoise, CPS) or milli handkerchief (mPas) is unit representation, and can be at about 5mPas within the scope of 3000mPas.The content of glycosamine be regarded as deacetylation degree (degree of deacetylation, DD) and can be in 30% to 95% scope.The chitosan that is crystal form is insoluble in the aqueous solution over pH7 conventionally, yet in diluted acid (pH<6.0), the protonated free amine group on glycosamine promotes the dissolving (Kim, Seo etc., 2008) of this molecule.In general, chitosan has the reactive functional groups of three types, is respectively amino and primary hydroxyl and secondary hydroxyl in C (2), C (3) and C (6) position.These groups allow chitosan to modify for application-specific, and it can prepare the various supports that are applicable to organizational project application.The chemical property of chitosan again for covalency and ion modification provide many may, thereby allow extensively to adjust machinery and biological nature.

chitin processing

As the above mentioned, chitin is present in numerous sorted group.Yet commercially available chitin is normally separated from ocean crustacean (as brown shrimp (shrimp)).The shell of crustacean is comprised of 30-40% protein, 30-50% calcium carbonate and 20-30% chitin, and the pigment that contains lipid in nature, as carotenoid (astaxanthin, ASX (astathin), canthaxanthin, phylloxanthin and beta-carotene).The ratio of these components is with changing in species and season.

When dissolving calcium carbonate by acid treatment, alkaline extraction makes protein denaturation and dissolves subsequently, and the step of decolouring is subsequently while extracting chitin, by removing astaxanthin, mainly obtains colourless to canescence product.This preparation method is a factor that affects sample characteristic.Early stage research is clearly confirmation, and the specific features of these products (Mw, DD) depends on process conditions.Yet, commercially available chitin typically the deproteinization by first step subsequently the demineraliting of second step prepare.Under these conditions, extract " chitin of disintegrate ", wherein chitin has lost natural structure.On the other hand, when there is demineraliting in first step, extract " chitin of compression ", wherein natural chain and filamentary structure are complete and stable.The chitosan of being prepared by arbitrary chitosan extracting method is all applicable to the present invention.In addition, the present invention does not limit the chitosan source from natural, semi-synthetic or synthetic source.

chitin deacetylation changes into as chitosan

Chitosan is to prepare by being hydrolyzed chitinous acetamido.The resistance of these groups of giving due to the substituent trans layout of C2-C3 in sugar ring, this is normally hydrolyzed and is processed (Horton and the Lineback1965) carrying out by caustic alkali.Conventionally need in strong alkali aqueous solution, to chitin, heat-treat to obtain the chitin (DD surpasses 70%) of part deacetylation, be called chitosan.Conventionally, at the lower working concentration of high temperature (100 ℃), be sodium hydroxide or the potassium hydroxide of 30-50% (w/v).This caustic hydroxide/by the use of thermal means has the effect that reduces or remove potential bacterial endotoxin simultaneously, and what this biomedicine for gained chitosan material was medical is useful.

The preparation method that depends on chitinous source and chitosan, chitosan DD can be within the scope of 56%-99% (Abou-Shoer2010).The factor that affects deacetylation degree comprises alkali concn, first pre-treatment, chitinous granularity and density.The maximum DD that in fact, can realize in single alkali treatment is about 75-85% (Roberts1998).In general, during deacetylation, condition must be to be suitable for making chitin deacetylation within reasonable time and obtaining the condition of chitosan, and this chitosan (subsequently) dissolves in acetic acid,diluted.Clearly, the primary factor of relevant chitosan fine structure is the chemical polydispersity (Roberts1998) of DD value.During chitosan deacetylation, there is the degraded of polymer chain.The chitosan stent with low DD value (75-85%) presents more regular structure, and hole quite evenly and with polygon transverse cross-section parallel (Tigli and Gumusderelioglu2008).For the support with high deacetylation degree (>85%), side direction pore communication is much lower.Also carry out the research of expanding, but do not found the relation between DD and expansion ratio.For the mechanical test demonstration of chitosan stent, under higher DD, mechanical strength is higher.The biodegradability of support also depends on DD.

the depolymerization of chitosan

The main limitation of using in some applications chitosan is its high viscosity and the low solubility under neutral pH.Low Mw chitosan and oligomer can be prepared by hydrating polymer chain.For some application-specific, have been found that these less molecules are more useful.Chitosan depolymerization can chemical mode, enzymatic mode or physics mode carry out.Chemical depolymerization is mainly the acid hydrolysis being undertaken by use HCl or passes through to use HNO ₂and H ₂o ₂the oxidation reaction of carrying out is carried out.Have been found that with regard to HNO ₂the amino of attacking deacetylation glucosamine units, makes adjacent glycosidic bond cracking subsequently, and the method is specific (Prashanth and Tharanathan2007).The in the situation that of enzymatic depolymerization, by preparing the low-molecular weight chitoglycan with highly-water-soluble as several enzymes such as chitinase, chitosanase, glucose enzyme and some protease.The known multiple non-specific enzyme that can make chitosan depolymerization, comprises lysozyme, cellulase, lipase, amylase and pectase.In this way, allow to carry out locational choice depolymerization (Aranaz, Mengibar etc., 2009) under temperate condition.

Between the Mw of chitosan and its expansion behavior, there are not dependency (Roldo, Hornof etc., 2004; El-Kamel, Ashri etc., 2007).Hot strength (tensile strength, TS), elongation at break percentage ratio (EB%) and elastic modelling quantity (elastic modulus, EM) are intensity and the elastic important parameters of indication thin film.Set up for evaluating the ASTM international standard method of testing (ASTM2002 of the physical parameter of thin film or film; ASTM2006).The chitosan film of medium Mw has the highest TS and EM value, is the chitosan film (El-Kamel, Ashri etc., 2007) of high Mw and low Mw subsequently.On the other hand, the chitosan film of low Mw obtains the highest EB%, is the chitosan film of high Mw and medium Mw subsequently.

the impact that hole changes

The mechanical property of the support based on chitosan depends on pore size and hole orientation.By the freezing and lyophilizing by chitosan solution, or by forming as methods such as " inner Bubbling method (internal bubbling process, IBP) " chitosan that is the loose structure that interconnects, in IBP method, by CaCO ₃add in chitosan solution, by using, be applicable to chitosan-CaCO that mould produces given shape ₃gel (Chow and Khor2000).For the extension test demonstration of hydrated sample, to compare with atresia chitosan film (5-7MPa), porous chitosan film has elastic modelling quantity (0.1-0.5MPa, wherein the MPa unit=N/mm significantly reducing ²).The extensibility of perforated membrane (maximum strain) is orientated and is being similar to the value of atresia chitosan (approximately 30%) to variation between surpassing 100% with pore size and hole.Perforated membrane shows the peculiar load-deformation curve of composite, has two completely different regions: the low modulus district under low strain and be converted to high 2-3 modulus doubly under high strain.It is reported, the hot strength of these loose structures is (Madihally and Matthew1999) within the scope of 30-60kPa.

Chen and Hwa have reported the molecular weight and the impact (Chen and Hwa1996) of its degree of crystallinity for chitosan film mechanical property of chitosan used.That is to say, owing to may there is entanglement difference, the molecular weight of chitosan used is lower, and the hot strength of prepared chitosan film is lower.In other words, use the chitosan of lower molecular weight to produce less entanglement.The degree of crystallinity difference of chitosan may be owing to another factor.The molecular weight of chitosan used is lower, and the enthalpy of gained film is less.These show, the lower hot strength of this film is lower the causing of degree of crystallinity in the chitosan film of being prepared by low-molecular weight chitoglycan.

biodegradability

Chitosan is not present in mammal, but can, in vivo by some enzymatic degradation, it should be noted that lysozyme, chitinase and NAG enzyme (Dalian, da Luz Moreira etc., 2007 most; Kim, Seo etc., 2008) (Aranaz, Mengibar etc., 2009) (Niekraszewicz2005).Biodegradation causes discharging the nontoxic oligosaccharide of different length, and these oligosaccharide can be incorporated into subsequently in glycosaminoglycans and glycoprotein, enter metabolic pathway or drained.Lysozyme, is present in mammalian tissues and relates to the non-specific glycoside hydrolase of natural immunity, seems the degraded of chitin and chitosan to play an important role.Degradation kinetics seems to be inverse relationship with crystallization degree, and crystallization degree is controlled by DD mainly.In addition, the distribution of acetyl group also affects biological degradability, because acetyl group does not exist or it is uniformly distributed (random but not block distributed) and causes extremely low enzymatic degradation speed.

Finally, some research reports, chain length (Mw) also affects degradation rate.About understanding and the control of the degradation rate of the material based on chitosan and medical treatment device merits attention especially, because degraded is absolutely necessary in many micromolecule and macromole discharge the regeneration application of application Zhong Ji functional organization.In some applications, the control that the speed of scaffold degradation should reflect the speed that new organization forms or be suitable for bioactive molecule (for example native compound, medicine, biological product, nucleic acid, vaccine and immunoeffectors) discharges.Therefore, understand and mechanism and the speed of controlling every kind of material degradation is very important.

Degradation rate also affects biocompatibility, because the degradation rate being exceedingly fast will discharge (and accumulation potentially) amino sugar, these sugar may cause slight inflammatory reaction.The chitosan sample with low DD can bring out the inflammatory reaction that acute degree is higher, and the chitosan sample with high DD brings out few reaction because degradation rate is low.Through showing, degraded can reduce and increase with DD.In other words, generally speaking, by increasing acetyl groupization, can promote degraded (Lim, Song etc., 2008).Kofuji etc., by observing the variation of chitosan solution viscosity under lysozyme exists, have studied the enzymatic behavior (Kofuji, Qian etc., 2005) of various chitosans.They find, the chitosan with low DD tends to more promptly degrade.Yet, other author's reports, the difference of degraded is that the distribution difference of acetamido in chitosan molecule causes.This is by due to the difference of deacetylation condition, and the difference of these conditions affects the viscosity of chitosan solution by changing repulsive force in intermolecular or molecule.Therefore, can reach a conclusion, only from DD, cannot estimate degradation rate.

biocompatibility

Chitosan demonstrates splendid biocompatibility, but this characteristic depends on the feature (for example, natural origin, preparation method, Mw and DD) of sample.Although digestion (oral cavity/gastrointestinal) enzyme is degrade chitosan partly, when Orally administered, it is not absorbed.For this reason, think that chitosan cannot supply biological utilisation by oral route.The LD50 of chitosan in mice is about 16g/kg, has high dosage and with insignificant acute toxicity.It is reported, the toxicity of chitosan depends on DD.The reports such as Schipper, DD shows hypotoxicity higher than 35% chitosan, and DD causes dose dependent toxicity (Schipper, Varum etc., 1996) lower than 35% (that is, chitin).On the other hand, the Mw of chitosan does not affect toxicity (Schipper, Varum etc., 1996).

With myocardial cell, endotheliocyte and epithelial cell, fibroblast, hepatocyte, chondrocyte and keratinocyte, proved in vitro the cell compatibility (Aranaz, Mengibar etc., 2009) of chitosan.It is relevant to the DD of sample that this characteristic seems.When the positive charge of this polymer increases, due to the existence of free amine group, the interaction between chitosan and cell also increases.Keratinocyte and fibroblast are depended on two factor: DD and cell type in the adhesiveness and the propagation that have on some chitosan films of different DD.In two kinds of cells, cell adhesion percentage ratio obviously depends on DD, with this parameter, increases.Cell type is also the factor that impact adheres to, and fibroblast is more favourable, and the negative charged surface that these cells represent is more than keratinocyte.On the other hand, by increasing DD, propagation obviously reduces.Therefore, in wound healing and biologic applications, the balance of cell adhesion and cell proliferation needs suitable DD.

The chitosan film that contains different Mw chitosans has different adhesions, but statistical analysis exposure, the bioadhesion power between thin film is without significant difference.On the contrary, the demonstrations such as Roldo, the low Mw chitosan of maximum desorption force rate of medium Mw chitosan and high Mw chitosan are wanted high (Roldo, Hornof etc., 2004).

Impure chitin and chitosan containing residual protein can cause allergic reaction in some individualities, as anaphylaxis.In sample, the content of protein depends on the source of sample and especially preparation method.For example, when as described above prepared by (, acid subsequently highly basic adds heat treatment), the chitosan of purification is non-sensitization.Although the crowd of 0.2-0.3% shows ocean crustacean irritated (Osterballe, Hansen etc., 2005; Osterballe, Mortz etc., 2009), but distinguished author Riccardo doctor Muzzarelli draws to draw a conclusion for chitosan:

It is unadvisable chitin being interpreted as to allergenic substance at present, need to carry out more clinical research and genetic research.Eriocheir sinensis, brown shrimp, prawn (prawn) and Lobster (lobster) chitin, once and the gradational chitosan of institute is through purification, just should not be regarded as " crustacean derivant ", because separable programming has been removed protein, fat and other pollutant to a certain extent, allow thus and be categorized as chemicals, no matter how it originates from.[(Muzzarelli2010) the 305th page]

Having identified main brown shrimp anaphylactogen is muscle protein tropomyosin ... the glycosamine in brown shrimp source is safe, even individual irritated to tropomyosin.Villacis etc. point out, the glycosamine complementary goods of being manufactured by various manufacturers should be containing the anaphylactogen of related levels [76] clinically.Gray etc. clearly point out, " shellfish shrimps allergy is that the IgE antibody by the antigen in shellfish shrimps meat rather than in shell causes; Therefore, taking glycosamine complementary goods should be safe for the patient of shellfish shrimps allergy " [77].[(Muzzarelli2010) the 300th page]

In addition, with regard to the chitosan of purification, as the material in " wound dressing " product, doctor Muzzarelli points out:

" in experiment and clinical front operation test, use chitin/chitosan and its derivant never to cause allergy or Other diseases." [(Muzzarelli2010) the 304th page]

the consideration of hemostasis

The Mw of chitosan also affects erythrocytic combination or coagulation (Mi, Shyu etc., 2001; Ishihara, Obara etc., 2006; Pang, Chen etc., 2007; Aranaz, Mengibar etc., 2009; Zhang, Xia etc., 2010).In recent paper, for solid-state chitosan and chitosan acetic acid normal saline solution, compare Journal of Sex Research (Jian, Feng etc., 2008).Tested Mw and be 2000 to 400kDa and DD be 90% to 70% some chitosan samples.Find that solid-state chitosan and " chitosan acetic acid normal saline solution " follow different hemostatic mechanisms.When blood is mixed with chitosan acetic acid normal saline solution, red cell agglutination and these cytomorphosiss.With at 100-1, the impact of the Mw between 000kDa in scope is compared, the DD in chitosan acetic acid normal saline, especially high DD, has appreciable impact for erythrocytic abnormal coagulation and distortion.Yet this phenomenon is not observed in solid-state chitosan.The solid-state chitosan with high DD is stronger in conjunction with more platelet and anastalsis.

The commercially available numerous medical device products that contain chitosan and its salt form all can be used for Bleeding control (for example, acid lyophilizing chitosan sponge).These devices are typically as wound dressing or " binder " and for the outer surface (referring to the device below with reference to document: FDA approval) of wound.

mucosal adhesive

Some factors can affect the mucosal adhesive of chitosan, as the physicochemical characteristics of physiological variable and chitosan.Mucosa forms by being called mucinous glycoprotein, owing to having salicylic acid residue, is rich in negative charge.At stomach, chitosan is positively charged due to sour environment, and therefore, it can interact by electrostatic force and mucin.The degree of this associating depends on the salicylic amount that exists in mucin and Mw and the DD of chitosan.Have been found that the permeability in mucin layer also increases when the Mw of chitosan increases, and make thus mucosal adhesive strengthen (Lehr, Bouwstra etc., 1992).On the other hand, higher DD causes molecule charge density to increase, and adhesion characteristics become more relevant (He, Davis etc., 1998).

antimicrobial acivity

An inherent character of chitosan is that it gives remarkable antibacterial activity (No, Park etc., 2002 for broad spectrum of bacteria; Jou, Yuan etc., 2007).Aimin etc. (Aimin, Chunlin etc., 1999) show, and chitosan can reduce in rabbit by the experimental myelitic infection rate bringing out of staphylococcus aureus (Staphylococcus aureus).This is relevant with the anion on bacteria cell wall with the cationic of being given chitosan by amino.Interaction between the chitosan of positively charged and electronegative microorganism wall causes spilling of cellular content.The synthetic inhibition of the combination of chitosan and DNA and mRNA be via chitosan penetrating in microbial cell solute and disturb the synthetic of mRNA and protein and (Liu, Guan etc., 2001) occur.

Other mechanism has also been proposed.Chitosan can, by serving as chelating agen, make metal, trace element or necessary nutrient with normal speed, grow to suppress growth of microorganism for confession organism.Chitosan can also interact with protein floculation, but this effect has high pH dependency.

In addition, chitosan also has antifungal property.Some authors propose, and chitosan can be explained by more directly upsetting film function for the anti-microbial effect of filamentous fungi.Yet the antimicrobial acivity of still not understanding chitosan is to be caused by growth inhibited effect (mycostasis) or cell death (fungicidal action).

antioxidant activity

Chitosan has demonstrated the remarkable removing ability for different free radical materials, and its result is suitable with the result obtaining with commercially available antioxidant.Based on removing 1, the bitter diazanyl (1 of 1-diphenyl-2-, 1-diphenyl-2-picrylhydrazyl, DPPH), the ability of hydroxyl, peroxide and alkyl diradical, the sample that is 90%, 75% and 50% to the DD being prepared by Carapax Eriocheir sinensis chitin is evaluated.Result discloses, and the chitosan with higher DD shows the highest removing active (Park, Je etc., 2004).On the other hand, the chitosan of analyzing Bu Tong size for superoxides and hydroxyl radical free radical with and sulfate-derivatives.Between the Mw of discovery chitosan and activity, be negative correlation.Chitosan sulfate derivant presents stronger clean-up effect for peroxide radical, but the minimum chitosan of Mw demonstrates than the significant ferrous ion chelating of other chitosan effect.The chelation of metal ion is that chitosan can be regarded as one of reason of the potential Natural antioxidant.Chitosan can, by the ferrous ion existing in chelating system, be eliminated its enzymatic oxidation activity or its delay lipid oxidation (Peng1998) that is converted to iron ion thus.

the current application of chitosan

Chitosan (natural cationic polysaccharide) and its salt form (for example, acetate, lactate, chloride, phosphate etc.) as being used for multiple application, especially the nontoxic and biodegradable biopolymer in food, medical treatment device, cosmetics and hair products and medicine, has caused sizable concern (Johnson and Nichols2000).

With regard to food, in recent years, chitosan is owing to being used to nonprescription drugs by bound fat, as the dietary supplement in multiple nutrients supplementary or cholesterol-lowering agent.Having identified chitosan is the general biological polymer of natural origin, due to its anti-microbial effect for food spoilage microorganism and anti-oxidation characteristics and for food antiseptic.PH dependent solubility can be used water treatment and be configured as various shapes (for example beadlet, thin film and film).Describe, beadlet and granule are applicable to resin, filler, absorbent, adsorbent and insulant (Smith1994) (Unger and Rohrbach1996).Existing lot of documents proves, uses chitosan coat can extend the storage characteristics of many fruits and vegetables as protective barrier.

the Medical application of chitosan structure

Chitosan is for example used to, in research and/or commercial product that wound healing management (, wound dressing and " binder "), implantable device system (as plastic surgery and periodontal complex, for the support of tissue regeneration) and drug delivery system unify in DNA delivery system because of its biological nature.

For many years, chitosan, as biodegradable natural biological copolymer, has been used as biocompatibility wound dressing.Material based on chitosan has high degree of biocompatibility, nontoxic and only have early stage, slight, a dominant inflammatory reaction of macrophage.In general, the uniqueness of chitosan chemistry and biological nature, biodegradation feature and biocompatibility arouse attention it in biomedical applications.The product of chitosan-containing is currently available on medical market, typically as for promoting the medical treatment device wound dressing of I class or " binder " of the US FDA of wound healing.The application in the world of product based on chitosan even may be more extensive than the U.S..

the safety of the chitosan of purification in human body

The safety of the chitosan of purification in human body be wide coverage (Illum1998; Baldrick2010).In various situations, confirmed the safety in human body:

1. the device of FDA approval: the chitosan of purification is the I class medical treatment of numerous US FDA approvals and the component of dental apparatus, and is in most of the cases as key component.It is to use with various finished product forms, as the film composition in granule, binder and gauze, and " sponge " of lyophilizing.The example of the I series products that FDA510 (k) Premarket Notification is listed comprises local hemostasis granule and the ChitoGauze in HemCon binder, the dressing of HemCon dentistry, the wound dressing of HemoHalt hemostatic adhesive bandage, the dressing of Aquanova superabsorbent, the solvable bag of CELOX.

2. gRAS food additive: chitosan is usingd various chitosan manufacturers (for example Primex) and " is certainly determined " that level is regarded as it is generally acknowledged safe (Generally Accepted as Safe, GRAS) as food additive.As far as we know, follow complete FDA and comment, the GRAS title under the higher level of " not adding comment " not yet occurred.Chitosan is thought and can be used safely in food by scientific circles, but has any should be noted: the chitosan of picked-up has affinity and can reduce gastrointestinal lipid absorption dietary lipids.

3. cosmetics and consumer's skin nursing products: chitosan is listed in the international name of cosmetic material (International Nomenclature of Cosmetic Ingredients, INCI).Chitosan and its various salt forms (for example lactate, glycollate, Ascorbate, formates and Salicylate) and other organic derivative are listed in for the composition of cosmetics and consumer's skin nursing products and are used by numerous suppliers.Yet chitosan is not yet assessed (Cosmetics Ingredients Review, CIR) by cosmetic material and is evaluated.For safety, this industry specialists group assessment the safety of the extremely limited cosmetic material of quantity.As far as we know, chitosan Wei Bei expert group takes into account, and scientific circles think that it is safe for consumer's skin nursing products and cosmetics.

organizational project

Organizational project is multi-disciplinary science, comprises the ultimate principle of attempting the biological substitution product of exploitation exhaustion tissue and organ in material engineering and molecule/cytobiology.In the most comprehensive meaning, the live body that organizational project attempts to manufacture health substitutes part.Langer and Vacanti (Langer and Vacanti1993) report is based on living cells, signaling molecule and polymer support for the most popular method of through engineering approaches biological substitution product.The synthetic neoblastic substrate of cell and replace ill or damaged tissues to work, and support to be cell can effectively realize its task (as adhered to, propagation and differentiation) provides applicable environment.The function of signaling molecule is help and promote cell regeneration new organization.Support not only provides the interim three-dimension-framework that forms designed tissue, but also provides the control of space-filling and biological activity signaling molecule to discharge.In order to carry out these different functions in organizational project, support should meet following requirement: (1) and the biocompatibility of tissue and the environment of promotion cell adhesion; (2) to form the iptimum speed of speed corresponding to new organization, carry out biodegradation; (3) nontoxic and non-immunogenicity; (4) best mechanical property; And (5) are suitable for transporting enough porositys and the form of gas, metabolite, nutrient and signaling molecule in support and between support and local environment.

Chitosan is one of the most promising biomaterial in organizational project, because it provides one group of distinct useful physical chemistry and biological nature, make it be applicable to the tissue regeneration of various types of other organ (as skin, skeleton, cartilage, liver, nerve and blood vessel).The regenerating tissues engineering research of carrying out has in the recent period proposed to support and the tissue of tissue damaged with support, because three dimensional matrix provides more favourable surrounding for cell behavior.Owing to having low immunogen activity, in check biological degradability and loose structure, chitosan bone is put up the material for the prospect that has much of design organization engineering system.

Known as the microstructures such as pore size, shape and distribution for cell invade, propagation and the function in organizational project have appreciable impact.The cell adhesion studies demonstration of carrying out on support, higher DD is conducive to cell adhesion (Seda Tigli, Karakecili etc., 2007).Yet the disclosure contains 56% to 99% chitosan DD.

The degradability of support is playing a key effect aspect the long-term behaviour of engineered cell/material construction body, because it has influence on many cell processes, comprises Growth of Cells, tissue regeneration and host response.If the organizational project by support for skeletal system, the degraded of support biomaterial should be relatively slow so, because it need to maintain mechanical strength, tissue regeneration completes or approached.Degradation rate also affects mechanical property and dissolution characteristics in time and inherently.

Recently, focus concentrates on by method of electrostatic spinning prepares polymeric nanofiber, and this is a unique technology, because depend on polymer and processing conditions, this technology can produce diameter at the several microns of chitosan nano fibers that arrive within the scope of tens nanometer.Electrostatic spinning applies high voltage to overcome surface tension of liquid to the capillary drop of polymer solution or melt, and can form thus the fiber meticulousr than conventional fibre spinning process.These nanofiber simulation n cell epimatrixs (extracellular matrix, ECM) 26S Proteasome Structure and Function, and in organizational project, as timbering material, recover, maintain or improve the function of tissue and cause great concern, because these materials have some useful characteristics, as high-specific surface area and high porosity.In the recent period attempted preparing the nanofibrous structures based on chitosan by electrostatic spinning, and obtained success in various degree.It is chitin and the chitosan nano fiber that 110nm and diameter (are measured by SEM graphical analysis) within the scope of 640nm at 40nm that Min etc. (Min, Lee etc., 2004) produce average diameter.Bhattarai etc. (Bhattarai, Edmondson etc., 2005) further infer, these nanofibers based on chitosan promote chondrocyte and osteoblast to adhere to and maintain characteristic cellular morphology.

wound healing

Chitin and chitosan make immunocyte and inflammatory cell (for example, PMN and macrophage), fibroblast and endothelial cell activation.These effects are relevant to the DD of sample, the effect that chitin presents than chitosan a little less than.Okamoto and colleague's report, chitosan affects all stages (Okamoto, Shibazaki etc., 1995) of repair in trauma in experimental animal model.In inflammation phase, chitosan has the unique haemostatic properties that is independent of normal coagulation cascade.In vivo, these polymer can also stimulate fibroblast proliferation and regulate the transfer behavior of neutrophil cell and macrophage, thereby improve follow-up repair process, as fibroplasia and re-epithelialization (Okamoto, Shibazaki etc., 1995; Kosaka, Kaneko etc., 1996).The reports such as Kosaka, the Cell binding of chitosan and cell activation characteristic are playing a key effect aspect its latent effect.These researchs have further confirmed that chitosan is suitable as wound healing material, wherein seed cells on the support based on chitosan bio-compatible and great-hearted engineered implant will be provided.

Chitosan oligomer also shows wound healing characteristic (Minagawa, Okamura etc., 2007).Propose, its wound healing characteristic is can stimulate fibroblast to produce by affecting fibroblast growth factor owing to it.Collagen protein subsequently produces the formation (Howling, Dettmar etc., 2001) that has further promoted connective tissue.

To chitin oligosaccharides the latent effect in wound healing with and for the ability of chronic intestinal illnesses, study (Deters, Petereit etc., 2008).The Wound Healing of chitosan oligomer and monomer merits attention especially, because lysozyme is degraded into these less molecules by chitosan polymer in vivo.

Have been found that implant based on chitosan causes minimum exotic reaction, have few or without fibre-coated.Typical agglutination is followed the formation of normal granulation tissue, conventionally follows angiogenesis to accelerate.Chitosan has the advantageous feature that promotes corium rapid regeneration and accelerated wound healing, is suitable for the multiple application from simple wound dressing to complicated artificial skin substrate.In the process of macrophage degrade chitosan implant, chitosan can stimulate anti-inflammatory cytokines cascade (Chellat, Grandjean-Laquerriere etc., 2005).

Desirable dressing for skin should be controlled the water evaporating in wound and lose with iptimum speed.The percutaneous moisture loss of normal skin (transepidermal water loss, TEWL) speed is 204g/m every day ², and the TEWL speed of the damaged skin of horny layer and epidermis injury can be at 279g/m every day ²(for " once " burn) is to 5138g/m every day ²in (for the granulation shape wound that lacks epidermis) scope.The water vapour permeability of wound dressing should prevent the accumulation of excessively dehydration and exudate.2500g/m every day recommending ²speed (in the intermediate range of damaged skin loss rate) will provide the moisture of proper level, simultaneously without the risk of wound dehydration.The time of depending on pervaporation (per-evaporation) before film cast, the moisture loss data of the asymmetric chitosan film of manufacturing are in every day 2109 to 2792g/m ²in scope (Mi, Shyu etc., 2001).The high porosity of cavernous transformation bottom increases the absorption of steam, and the thickness of fine and close skin layer reduces to make the diffusion of hydrone to increase, and causes thus the increase of moisture vapor transfer speed.

drug delivery system

The important application of chitosan in industry is developing drugs delivery system, as nanoparticle, hydrogel, microsphere, thin film and tablet.Owing to having cationic, chitosan can react with polyanionic, obtains PEC.Medicinal application comprises per nasal, eye, mouth, vagina, parenteral and transdermal drug delivery.Need three principal characters of the chitosan of consideration to be: Mw, DD and purity.When chitosan chain shortens (low Mw chitosan), these chains can directly be dissolved in the water, this is specially adapted to particular organisms medical application, when pH is maintained at about 7.0, or is specially adapted to dermatological or consumer's skin care applications when lower slightly (about 5.5-6.5).

In drug delivery, select the desirable chitosan type with some feature to can be used for developing lasting drug delivery system, prolong drug active duration, improves therapeutic efficiency and reduces side effect.The physicochemical characteristic of chitosan is for selecting suitable chitosan as the vectorial material particular importance of drug delivery.

Owing to controlling the variation of the loading of glycan substrate and the hydrophobic interaction of release characteristic, therefore DD controls degree of crystallinity and hydrophobicity (Draget1996) in chitosan.Zhang etc. also report, have shown that high chitosan DD and narrow polymer Mw distribute for controlling particle size distribution most important (Zhang, Oh etc., 2004).

Desai and Park observe, when the chitosan Mw for the preparation of microsphere increases, and ascorbic rate of release step-down (Desai and Park2006).They have studied release dynamics and have found that it follows Fick's law of diffusion (Fick's law of diffusion).

With regard to release in vitro research, the medication amount that the thin film that contains low Mw and medium Mw chitosan discharges is similar, but the thin film of preparing with high Mw chitosan is lower.Consider the direct relation between the molal weight of chitosan and the viscosity of its solution, this characteristic is predictable.By increasing the viscosity of polymer, make medicine pass formed gel layer to the diffusion delays (El-Kamel, Ashri etc., 2007) in release medium.

gene delivery

Due to positive charge, chitosan can interact with electronegative molecule (as DNA).This characteristic is used to prepare the non-virus carrier (MacLaughlin, Mumper etc., 1998) of genes delivery system first in nineteen ninety-five.Compare with viral vector, use chitosan to provide some benefits as the non-virus carrier of gene delivery.Main, chitosan does not produce endogenous restructuring, carcinogenesis and only causes slight immunoreation.In addition, chitosan/plasmid dna complex compound can easily be prepared with low cost.

The Mw of chitosan is a key parameter in the preparation of chitosan/DNA complex, because transfection efficiency is obviously relevant to chitosan Mw.High molecular weight chitosan produces very stable complex, but transfection efficiency is extremely low.In order to improve transfection efficiency, recent research has checked low Mw chitosan and the application of oligomer in gene delivery vector.In order to obtain high-level transfection, seem to realize the fine equilibrium between solution packing (better under low Mw) in extracellular DNA protection (better under high Mw) and effective cell.Lavertu etc. have studied the combination of several chitosans Mw and DD, find that use has 10kDa and has high transfection efficiency (Lavertu, Methot etc., 2006) with two chitosans combinations of 92%DD and 80%DD respectively.

Kiang etc. have studied in chitosan-DNA nanoparticles chitosan deacetylation degree to the impact of efficiency gene transfection (Kiang, Wen etc., 2004).The chitosan of height deacetylation (surpassing 80%) released dna is extremely slow.They propose, and use DD can promote the release of DNA lower than 80% chitosan because this Chitosan Reducing charge density, can increase the steric hindrance of the complex forming with DNA, and known its can accelerated degradation speed.Their report, when DD is reduced to 70% from 90%, luciferase reporter gene express to increase.The luciferase transgene expression that the formulation of 62% and 70% deacetylation causes is than high two orders of magnitude of the chitosan of 90% deacetylation.

chitosan film

Potential and the practical application of chitosan film or thin film is at intra-operative chorista layer as Obstruct membrane.Typically use three kinds of methods to prepare and there is low-density to highdensity membranaceous or film like chitosan structure.These preparation methoies are solvent cast, are separated and the conversion of submergence-precipitated phase (Madihally and Matthew1999; Hong, Wei etc., 2007).For whole three kinds of methods, for example, for example, by appropriate chitosan powder (, 75-90%DD/400-500mPas) is dissolved in and prepares variable concentrations (, chitosan solution 2-4%w/v) in 1% (v/v) acetic acid solution.Next, chitosan solution is poured in the silicone molds chamber of customization.With regard to this point, these three kinds of diverse ways (as described below) differ from one another.

In phase disengagement method, the acid chitosan solution of cast freeze overnight at-20 ℃, then 10 * 10 ^-3lyophilization 48 hours at-40 ℃ under millibar.Then, make the cryodesiccated chitosan material demoulding and process 4 hours so that chitosan polymer network stabilization is used distilled water cyclic washing with 1N NaOH, then put into 50 ℃ of baking ovens dry.Phase disengagement method produces relatively low-density porous " sponge ", and pore size can be controlled (Mi, Shyu etc., 2001) (No etc., 2002).

Freezing chitosan solution produces two or more distinct phases-typically water-cooled is frozen into ice, and chitosan biomaterial is displaced in independent solid phase.Need another step to remove freezing solvent (typically being ice), and make thus low density porous sponge, this is the common type of wound dressing.This method can be because of lyophilization (that is, lyophilizing) and/or freezing substitution step and is destroyed filamentary structure.

For solvent cast method, in 50 ℃ of baking ovens, the acid chitosan solution of cast is dried to remove solvent simply, thereby leaves chitosan film.After dry, with 1NNaOH, process chitosan film 4 hours, with distilled water cyclic washing, to remove the reactant of any trace, then put into 50 ℃ of baking ovens dry.After with the method cast solution, when solvent starts to vaporize, the vaporization of the solvent of the lip-deep ratio of solvent of polymer solution inside is fast, polymer concentration is increased sharply, thereby form stratiform by means of gluey particle.After forming surface layer, the vaporization of solvent is slowed down.The dissolubility deficiency of chitosan is so that system held homogeneous solution form, and causes and be separated.From the solvent of this homogeneous solution separation, form the phase that lacks polymer, the encirclement mutually of being rich in polymer.The exchange of acid flux material and neutrality alkali makes polymer network stable.

In the third method,, submergence-precipitated phase transforms (immersion-precipitation phase inversion, IPPI) in method, in 50 ℃ of baking ovens, make the acid chitosan solution (partly) of cast dewater 1 hour to form asymmetric membrane, by being immersed, within 0.2MNaOH solution 24 hours, make the chitosan polymer that is form membrane stablize subsequently.Then use deionized water cyclic washing gained film, and follow lyophilization 48 hours.IPPI method produces the asymmetric porous membrane with three layers: then fine and close skin, not too fine and close intermediate layer are spongy porous layers, are all (Hong, Wei etc., 2007) that can control.

About the commentary of chitosan and its purposes open (Kato, Onishi etc., 2003; Niekraszewicz2005; Boateng, Matthews etc., 2008; Aranaz, Mengibar etc., 2009; Zhang, Xia etc., 2010).

The preparation of chitosan sponge and purposes are described in prior art:

(1) neutral sponge (Zhang, Cheng etc., 2006 of relevant unpressed lyophilizing; SedaTigli, Karakecili etc., 2007; Blan and Birla2008); And

(2) non-neutral sponge (Tully-Dartez, Cardenas etc., 2010 of relevant unpressed lyophilizing; McAdams, Block etc., 2011).

Also having described some other increases the method for the density of chitosan material, comprising:

(1) the acid sponge of lyophilizing is compressed to unspecified density (McCarthy, Gregory etc., 2008; Gregory and McCarthy2009);

(2) the acid sponge of lyophilizing is compressed to specified density (McCarthy, Gregory etc., 2008 that are less than or equal to 0.8g/cm3; Gregory and McCarthy2010; McAdams, Block etc., 2011; McCarthy, Gregory etc., 2011);

(3) asymmetric air drying (Ma, Wang etc., 2001; Thein-Han and Stevens2004; Kuo2005; Kuo, Chang etc., 2006; Dallan, da Luz Moreira etc., 2007; Duan, Park etc., 2007; Hong, Wei etc., 2007; Pang, Chen etc., 2007; Kuo2008) (Ma etc., 2001) (Duan etc., 2007); And

(4) electrostatic spinning rolling subsequently (Yeo, Jeon etc., 2005; Li and Hsieh2006; Park, Kang etc., 2006).Electrostatic spinning produces thinner neutral chitin fiber, and these fibers can blended togetherly become layered network product.Electrostatic spinning technique is not suitable for the present invention described herein.

Can come stiffened shell glycan structures (Masuoka, Ishihara etc., 2005 by carrying out chemical crosslinking at needs or do not need photoactivation in the situation that; Obara, Ishihara etc., 2005).Yet these cross-linking methods all can not be increased to the density of chitosan high density scope of the present invention described herein.

Asymmetric air drying increases the density of chitosan solution by the surface evaporation that acid flux material is exposed from chitosan solution.When removing solvent, on the surface of exposure, the density of chitosan increases.The method of this increase chitosan density can produce fine and close membranaceous chitosan device.The inhomogeneities that specific question is the surface evaporation of solution in mould that profit exists in this way, and the density that may reach in not compression situation is limited.Only by another problem that air drying is manufactured fine and close chitosan film structure, be, the excessive expansion when moistening of dry film, and have clinically problem for the material that is intended as fine and close barrier film.Therefore, be different from prior art, the invention describes the new method of manufacturing the membranaceous chitosan material of high density, these methods have solved current problem.

General introduction

So far, in operation and wound healing application, use the chitosan of purification as Obstruct membrane, to be subject to the restriction of chitosan physical characteristic.As the chitosan of preparing described in prior art can produce thin film, film or sponge, it has density or other physical characteristic being not enough to for medical application.When what typically need according to medical application, when moistening reaches flexible degree, prepared density <0.6mg/cm ³firm stitching and the operation of the undercapacity that has of chitosan during settling with buttressing operation reliably.Therefore, developed and manufactured density >0.6mg/cm ³the new method simultaneously with the chitosan film of other beneficial characteristics.This high density chitosan thin film or film provide and have been applied to reliably clinical necessary intensity and operating characteristics.Specifically, high density chitosan thin film of the present invention or film have be applicable to medical domain superior tensile strength, sew up to retain (, resistance to pull out suture), elasticity, applicable thickness and shape memory are (, and when rehydrated, still have a limited expansion adaptability).

Increase in some methods of chitosan density becoming known for, most popular method comprises the compression of the chitosan sponge of lyophilizing.Although can use enough pressure that the chitosan stent of lyophilizing is compressed into high density, the shortcoming of this common programs is, the lyophilizing support of compression keeps shape memory after rewetting, and excessively recoils into clinically as the unacceptable thickness of film.Method by this compression lyophilizing sponge can not limit the thickness of recoiling after moistening and in membrane structure, maintain enough density, and is therefore unsuitable for preparing the dense film that intensity is enough to be used in clinical manipulation and stitching.Therefore, the invention describes the about 0.6-1.6g/cm of preparation ³and thickness is less than the new method of the fine and close chitosan film of 2mm, and the present invention has removed the general procedure of manufacturing sponge before compression.

Essence of the present invention is preparation and the purposes of membranaceous chitosan material, this material is finer and close than the chitosan sponge of lyophilizing, and there is other characteristic, as (suitable hot strength, stitching retain, resistance to pull out suture), elasticity and enough shape memory are (, adaptability), and when rehydrated, still have limited expansion, these are obviously different from previously described chitosan material.The present invention does not limit from the chitin in natural, semi-synthetic or synthetic source or chitosan source.

Producing committed step of the present invention is:

1) acid chitosan solution is immersed in highly basic, until all acid in this material are all neutralized, and the chitosan gel rubber that gained solidifies has alkaline pH.The present invention carries out air drying before being not precluded within neutralization.In order to maintain the desirable chitosan concentration of per unit area, about 0.3-0.5g chitosan solution/cm preferably ², during described N-process, it is preferred with mould or model, retaining chitosan solution.Before described neutralization, preferably in mould freezing chitosan solution so that in the concentration stabilize of per unit area chitosan during N-process and promote to get rid of polymer before neutralization.At chitosan, from surface losses and before changing even chitosan structure, preferably use the habitual highly basic (being preferably the sodium hydroxide of 1-2 molar concentration) of technical staff of chemical field to make freezing chitosan float from the outside of freezing chitosan float to interior polymeric.

2) remove water or the liquid in solid chitosan gel, simultaneously compression shell polysaccharide.The dehydration preferably highly basic in the chitosan gel rubber solidifying and aqueous buffer solution or water exchange is carried out afterwards.Dehydration is preferably carried out in a vacuum under heat exists.Dehydration preferably in the presence of heat time, for example, reduces solvent phase (for example, water, aqueous buffer solution) by semipermeable membrane (, cellophane (Cellophane) or similarly fibrous material) and carries out under vacuum.Compression is preferably undertaken by the minimum line pressure that is scattered in equably 25 inches of Hg on chitosan gel rubber.

An important unique aspect of the present invention is by compression and dehydration combination, makes thus gel dewater between compression period.Another unique aspect of the present invention is that the pH of gel is neutral or alkaline during dewatering.

From the commentary of the following specific embodiment and by with reference to claims, can more clearly understand and understand these and other objects of the present invention, feature and benefit.

Describe in detail

Based on aforementioned discovery, provide density to be greater than 0.6g/cm herein ³new chitosan structure, prepare the method for compositions and use said composition for the method for described medical application under literature background.The method of preparing chitosan structure can characterize by following three consecutive steps:

A) provide the acid solution of water and chitosan;

B) neutralize described solution to form the gel of polymerization chitosan;

C) this polymerization chitosan gel rubber is dewatered simultaneously and compressed.

In a preferred embodiment, the density of gained high density chitosan thin film or film composition is greater than 0.6g/cm ³, and more preferably greater than 0.8g/cm ³.

In a preferred embodiment of the invention, the chitosan initial substance using in acid solution is about 70-95%DD.Yet the present invention also allows the DD of 56%-99%.

In a preferred embodiment, chitosan is to exist with chitosan alkali form.Yet, this chitosan also can be salt form, as chitosan acetate, chitosan succinate, chitosan adipate, Sea Cure CL 313, chitosan glutamate salt, chitosan lactate, chitosan aspartate, chitosan pyruvate, chitosan phosphate, chitosan glycollate, chitosan Ascorbate, chitosan Salicylate, chitosan formates or chitosan malate.

In another preferred embodiment of the present invention, chitosan initial substance has the average viscosity of about 400-500 centipoise (CPS) or milli handkerchief (mPas).Yet approximately 5 to 3000mPas chitosan initial substance viscosity is contained in the present invention.

In a preferred embodiment of the invention, chitosan is dissolved in 1% acetic acid.Yet the present invention also considers that acid flux material except acetic acid and percentage of solvents are within the scope of 0.1%-10%.For instance, pH, lower than 5.0 suitable organic acid, as formic acid, glycolic, citric acid or lactic acid, is also applicable to.Other applicable acid comprises hydrochloric acid, glutamic acid, aspartic acid, ascorbic acid, acetone acid, malic acid, maleic acid, fumaric acid, glucuronic acid, sorbic acid and folic acid.

In a preferred embodiment of the invention, in this solution, the concentration of chitosan is 2%-4%.Yet 0.1% to 25% chitosan concentration is contained in the present invention.

In another preferred embodiment of the present invention, before forming neutral chitosan gel rubber, chitosan is dissolved in acid flux material, keep 7 days (before neutralization, having or do not exist freezing step).Yet the present invention also considers before being about to form neutral chitosan gel rubber or reaches chitosan solution prepared in 2 years (having or do not exist freezing step before neutralization).

In a preferred embodiment of the invention, a certain amount of chitosan solution is poured in a model or mould, reached the thickness of the about 0.3-0.5g chitosan solution of area of every square centimeter of this model or mould.Yet the present invention is encompassed in and is low to moderate 0.1g/cm2 before freezing in mould or model or up to the chitosan solution amount of 10g/cm2.

In a preferred embodiment of the invention, by this mould or model, this solution being applied to vibration makes chitosan solution degassed.Time of vibration is preferably 10 minutes.Yet the time of vibration of 1 second to 10 days is contained in the present invention.In alternate embodiment, the present invention is contained by applying vacuum makes chitosan solution degassed.

In a preferred embodiment of the invention, in mould or model freezing chitosan solution so that it becomes the chitosan float of solidifying.In a preferred embodiment of the invention, freezing chitosan solution 1 hour at approximately-80 ℃.In alternate embodiment, at approximately-20 ℃, freezing chitosan solution is 16 hours.Yet the present invention is encompassed in freezing chitosan solution at the temperature within the scope of 0 ℃ to-276 ℃, continue the time that is enough to make chitosan solution freezing from 1 minute to 365 days.The present invention is also encompassed in this one-phase of the method the not possibility of freezing chitosan solution.

In a preferred embodiment of the invention, make (if freezing) chitosan float of solidifying with the solid form demoulding (shifting out from mould), with solid form, immerse in alkali (as 1-2M sodium hydroxide) subsequently, keep 24 hours to neutralize the acid flux material in the chitosan float of solidifying completely, thereby produce polymeric gel.Yet concentration and the volume of the required alkali of the acid flux material in the chitosan gel rubber that solidifies of neutralization completely, and the persistent period of submergence, can change according to size and the acidity of the chitosan float of solidifying.Any in chemical field several alkali known to the skilled contained in the present invention, as sodium hydroxide or potassium hydroxide, concentration at 0.1M within the scope of 10M, and Immersion time 1 minute by 3 months within the scope of.Can use substituting hydroxide, and it comprises calcium hydroxide and magnesium hydroxide.

In a preferred embodiment of the invention, in deionized water or distilled water or aqueous buffer solution washing have alkaline pH neutral chitosan gel rubber 24 hours so that alkaline solution become neutrality or be substantially neutral pH (for example, pH5-11,5-9 or 5.5-7.5).Yet the present invention is contained from the wash time of 1 minute to 3 months.The present invention is also encompassed in deionized water or distilled water or the aqueous buffer solution that uses continuous-flow during this rinsing step.The present invention is also contained and is not washed neutral chitosan gel rubber.

In a critical aspects of the present invention, from neutral chitosan gel rubber, remove liquid, compress this chitosan simultaneously.Dehydration preferably utilizes vacuum and heat to carry out.That compression is preferably carried out with the minimum line pressure of 25 inches of Hg and be preferably scattered in equably on chitosan gel rubber with acquisition uniform films.Yet, contain the compression of using the minimum line pressure of 5-500 inch, 10 to 100 inches or 20 to 50 inches Hg to carry out.Dehydration and compression are preferably carried out at 80 ℃ of temperature.Yet the present invention is encompassed in and at the temperature within the scope of 2 ℃ to 150 ℃, 40 ℃ to 120 ℃ or 50 ℃ to 100 ℃, chitosan gel rubber is dewatered and compress.

Certainly, should be appreciated that, in being called the process of degasification, dehydration and physical compression can occur voluntarily or occur by heating under vacuum.The vacuum applying is preferably lower than atmospheric pressure, and is low to moderate 0.6,0.4 or 0.2 atmospheric pressure.

Dehydration and compression are preferably carried out the time of 4 hours.Yet the present invention is contained and is dewatered and be compressed in the time within the scope of 1 minute to 3 months.

In a preferred embodiment of the invention, applying before vacuum dewaters, neutral chitosan gel rubber is being placed on semipermeable membrane or inner.This semipermeable membrane promotes steam to scatter and disappear under vacuum subsequently, the integrity of the polymerization chitosan that maintenance is being dewatered and dewatered simultaneously.The alternative infiltration water of semipermeable membrane retains molded chitosan gel rubber simultaneously, and can be cellophane or other fibrous membrane or another material in the edge of semipermeable membrane or border.The high density chitosan thin film of dehydration or film can shift out from the semipermeable membrane using during dehydration subsequently.

In another preferred embodiment of the present invention, neutral polymerization chitosan gel rubber is immersed in glycerite, remain on the time within the scope of 1 second to 10 days, then put it on semipermeable membrane or vacuum dehydration is carried out in inside.In addition, this glycerite preferably in water or in aqueous buffer solution containing having an appointment 5% to 20% or 10% glycerol.Yet the present invention also can use the glycerol concentration in 1% to 50% scope during this process.

Gained chitosan structure is preferably thickness and is less than 10mm, 5mm, 2mm, 1mm or the even thin film of 0.5mm or the form of film.As discussed previously, the density of this structure preferably surpasses 0.6g/cm ³, and can be up to 1.6g/cm ³.In another preferred embodiment, the density of this structure surpasses 0.8g/cm ³, and can be up to 1.6g/cm ³.This thin film or film also can characterize by its pH, and this pH is preferably in 5.0 to 9.5 scope.This thin film or film can be cut open or grind and use with particulate form, but for example, due to its good physical characteristic (hot strength, elasticity and the resistance to pull out suture), preferably with thin film or form membrane, use.

In a preferred embodiment, chitosan film of the present invention or film do not need chemistry or photoinduction cross-linking step, and still obtain >0.6g/cm ³and more preferably >0.8g/cm ³dehydration density.Yet, for some application, comprise that chemistry or photoinduction cross-linking step may provide some benefits, as reduced biodegradation probability.

In another embodiment, the physical characteristic that gained fine and close chitosan structure of the present invention has is of value to the biomedical programs for animal, mammal or people, as the thin film of Operation or film.During when assessment hot strength, elasticity and/or to the resistance of pull out suture, fine and close chitosan material presents good physical features.Set up for evaluating the ASTM International Standards Method (ASTM2002 of these physical parameters of thin film or film; ASTM2006).These have the standard method of minor modifications, and (for example,, for extension test, according to ASTM standard method D1708-06a, band is semicircle but not half elliptic die plate pattern, and minimum widith is about 2.5mm (ASTM2006); For pull out suture, the width of band is about 5mm) be used to characterize gained fine and close chitosan structure of the present invention.

The invention discloses compositions, said composition comprises density and is greater than 0.6g/cm ³and be more preferably greater than 0.8g/cm ³the chitosan that is thin film or form membrane.In another preferred embodiment, the pH of this chitosan composite is 5.0 to 9.5.In another preferred embodiment, this chitosan composite comprises glycerol.

Finally, the invention provides the method for using structure of the present invention to treat, and therefore can be defined as Therapeutic Method, the method comprises: provide density to be greater than 0.6g/cm ³chitosan composite; And described compositions is positioned on animal body or in its body.In preferred embodiments, this animal is mammal or people, and in another preferred embodiment, before use, this structure is in water or aqueous buffer solution and in existence or do not exist one or more situations that are selected from the compound of medicine, biological agent, nucleic acid, vaccine, immunoeffectors or its salt to issue raw hydration.In another preferred embodiment, this chitosan composite as physical barrier thin film or Obstruct membrane with each organized layer in animal body separately.In another preferred embodiment, thin film or film on this animal, mammal or human body or in its body absorb in time and again, and its speed depends in part on DD and the thickness of this material.In another preferred embodiment, this chitosan composite is used as infection physics barrier film or Obstruct membrane on animal body or in its body.In another preferred embodiment of the present invention, the micromolecule in this chitosan film or film water permeable or aqueous solution.In another preferred embodiment of the present invention, multiple physical characteristic (for example, hot strength, elasticity and the resistance to pull out suture) promoted the fine and close chitosan film of gained or the good ease for use of film in animal, mammal or people's clinical setting separately or for example, with the combination of clinical manipulation feature (, wettability, for the adaptability at Operation position and the property sewed up).

Embodiment

Before neutralization in the method for optimizing of freezing chitosan solution, at-80 ℃, in mould by about 0.3-0.5g/cm ²chitosan solution cryotherapy within one hour, finally cause chitosan polymerization on the top surface exposing, when by microscope or scanning electronic microscope examination, in this surface, there is the fibrous porous structure of braiding.The gross density >0.6g/cm of gained dehydration film or film ³and more preferably >0.8g/cm ³, and slightly some is asymmetric, in relative bottom side, have more smooth, not too as fibriilar surface.

Before neutralization, in the method for optimizing of freezing chitosan solution, the physics that cryotherapy obvious super (for example, two hours) after an hour can cause freezing chitosan gel rubber and final membrane structure at-80 ℃ is cracked.

In there is freezing method for optimizing, before neutralization, in mould, at-20 ℃, freezing chitosan solution reduces the scope of braiding structure in final membrane structure.No matter cryogenic temperature how, the density >0.6g/cm of gained film ³.

Before neutralization, not freezing chitosan solution in the situation that, gained does not have visible braiding fibrillar structure through the film of overcompression and dehydration.Freezing regardless of existing or not existing, the density >0.6g/cm of gained film ³.

It is associated by by utilizing and not utilizing the mechanical property of the material producing in refrigerating process situation further to illustrate that before neutralizing acid, freezing acid chitosan solution adds compression with dehydration.When using Instron testing machine to measure the resistance to pull out suture, do not utilize the chitosan film of preparing in refrigerating process situation to there is the poor extraction power of every millimeter of film thickness 2.0N ± 0.3N, and before neutralization, under-80 ℃ of cryogenic temperatures, keeping 1 hour prepared film with same composition to there is the good resistance to pull out suture of every millimeter of film thickness 4.5N ± 0.1N.

In order to illustrate before dehydration and compression, with alkali, freezing chitosan float is neutralized into the importance of semi-solid gel, relevant trial failure of evaporating in the acid chitosan solution of compression.Dehydration adds compression needs semipermeable membrane (for example cellophane) to retain solute (chitosan polymer), allows solvent to pass through simultaneously, and wherein, in dry, the density of chitosan increases gradually.The possible reason that cannot dewater to acid chitosan solution by semipermeable membrane is that the not polymerization chitosan of viscous is finally accumulated in film surface and has stoped passing through of solvent.Result is to dewater to this solution, even under heat exists.For the same reason, after freezing acid chitosan solution, also cannot dewater immediately and compress.For the same reason, also cannot to moist acid chitosan sponge (by prepared by the lyophilizing of acid chitosan float), dewatering add compression, thus cannot be dried by vacuum dehydration.For the same reason, also cannot dewater and add compression moist, air dried acid chitosan structure.Before vacuum compression, by with in alkali and to make the polymerization of chitosan float be necessary.

In order to illustrate the importance that evaporates and compress neutral chitosan gel rubber, in the trial aspect the acid sponge of lyophilizing of compression drying, cause that chitosan film structure is cracked.The dry compressive films of moistening causes unacceptable recoil to be expanded and the not loss of polymeric membrane structure.

When neutral chitosan gel rubber being put into the acid solution of pH2.9, in the time of 20 hours, cause that chitosan gel rubber structure disintegrates, make chitosan structure loss, for example understand and after chitosan solution aggregates into gel in highly basic, maintain pH higher than 5.0 importance.

In pH4 or lower sour environment, after 24 hours, fine and close chitosan film or membrane structure are lost completely, for example understand after neutral chitosan gel rubber dehydration and maintain the importance higher than the pH environment of pH5.0.

In order to illustrate, make neutral chitosan gel rubber but not the sponge dehydration of lyophilizing and the importance of compression compresss generation 0.38g/cm to the neutral lyophilizing chitosan sponge of moistening ³not enough chitosan density.Dry neutral lyophilizing chitosan sponge is compressed and causes not enough chitosan density (0.065g/cm ³).

In order to illustrate the importance that dewaters and compress simultaneously, between compression period, in the situation that not carrying out suitably dehydration, providing the experiment of compression to produce cracking and not satisfied chitosan final structure.

Neutralization before vibration on the impact of acid chitosan solution on final membrane structure without impact.

In order to illustrate the biological relatedness with highdensity chitosan film or film, these films represent micromolecular permeability.For instance, use Franz pool technology, methylene blue and crystal violet (being respectively Mw285 and 373) in the high density chitosan thin film of being prepared by 4% chitosan solution or the permeable phosphate buffered saline of film (phosphate buffered saline, PBS) solution.This has confirmed that these thin film or film are to selected micromolecular permeability.Permeability for the nutrient on animal, mammal or human body or in its body may have physiological benefit.

For further illustrating the biological relatedness with highdensity chitosan film or film, work mammalian cell is inoculated on these films and in culture and maintains at least 3 days.Observe Cell binding and cell compatibility on these film both sides.Also confirmed the evidence of propagation and cell migration.As measured by microscopic image analysis, with respect to porous surface comparatively (that is, the top when in mould), keratinocyte migration is the most obvious on more smooth surface (that is, the bottom when in mould).These results provide the evidence of biocompatibility in vitro.

In order further to illustrate the biological relatedness with highdensity chitosan film or film, when these films being put into by operation to the place, bottom of the ulcer that in the maxillary of rat oral cavity, full-thickness operation is brought out, observe wound healing in mammal model.Healing is relevant with the re-epithelialization of ulcer to growing again of collagen protein in upper sub-endothelial matrix.The histologic analysis carrying out after film is implanted 1 to 12 week is also indicated, and high density chitosan is bio-compatible, and the while is also biodegradable or can be resorbent.On animal, mammal or human body or in its body, produce resorbent physical barriers in time and there is clinical efficacy.For instance, for example, physical barriers between different tissues (, skeleton contrast soft tissue) can promote the difference Healing Rate on the opposite side of this thin film or film.In addition, with regard to vitro enzyme degradation rate, expect similarly in body that again absorption rate (vide infra) depends on DD percentage ratio and/or the thickness of fine and close chitosan film or film.In other words, degradation rate can carry out " control " by changing DD percentage ratio and/or thickness at least partly.

In order to illustrate the associated of density and clinical function and effectiveness, between the density of dry chitosan film or film and other physical characteristic (as hot strength), there is strong correlation.With the ASTM standard method with minor modifications, (for example,, for extension test, according to ASTM standard method D1708-06a, band is semicircle but not half elliptic die plate pattern, and minimum widith is about 2.5mm (ASTM2006); For pull out suture, the width of band is about 5mm) be used to characterize the fine and close chitosan film of gained or film.By fine and close chitosan film that herein prepared by claimed method or film, demonstrate the directly related property between film density and hot strength.In general, fine and close chitosan film of the present invention or film in a series of experimental variablies that change with manufacture batch of material (are for example worked as, the amount of initial chitosan solution, be less than 1mm and typically be 0.2 to the build of 0.6mm, 70% to 95% DD percentage ratio, from different suppliers' raw material, while testing dry post processing (if any) etc.), in the test of using Instron testing machine, obtain following typical physical characteristic range: (a) approximately 2 to 14N maximum tension load (about 2.5mm minimum widith); (b) approximately 20 to 140MPa maximum tensile stress (about 2.5mm minimum widith); And (c) approximately 0.5 to the pull out suture maximum load (about 5mm width) of 4.5N.

In addition, what illustrate fine and close chitosan film of the present invention or the physical features of film and the dependency of clinical efficacy is density, hot strength, elasticity and the combination to the resistance of pull out suture, and wherein having some or all is all the desirable feature of implantable surgical membrane that can sew up.

For the method about disclosed herein and claimed illustrates the association of chitosan deacetylation; degraded at 37 ℃ in the dense buffering lysozyme soln of pH6.5 completes in 8 days for 70%DD film; for 75%DD film, in 11 days, complete; for 80% and 85%DD film part in 18 days complete, and still not obvious after 3 weeks under these conditions in 90% and 95% film.These result indications, starting polymerization material (that is, the chitosan powder that DD percentage ratio is different) is not destroyed by method of the present invention for the intrinsic sensitivity of external enzymatic degradation, produces fine and close chitosan film or film simultaneously.In addition, fine and close chitosan film of the present invention or film in without enzymatic degradation effect situation, keep the unstability to acid depolymerization (and solubilization) when putting into acetic acid solution or pH4 or lower buffer solution.

In order to illustrate before dehydration, for example, with glycerite (10% or 50% glycerine water solution), process the association of neutral polymerization chitosan gel rubber, gained thin film or film have and thin film or the similar high density of film not having to manufacture in this glycerite step situation, and there is useful high tensile, the resistance to pull out suture, and performance characteristic, as flexible and be easy to cutting.This combinations of attributes (that is, physical characteristic and clinical manipulation feature) provides the thin film with very big effectiveness or the membrane material that are applicable on animal, mammal or human body or in its body.

Various publications have been mentioned in the application in the whole text.The disclosure of these publications is all incorporated to accordingly by reference, to describe more fully the prior art level in the affiliated field of the present invention.It is evident that for those skilled in the art, in the situation that not deviating from scope of the present invention or spirit, can carry out various modifications and changes to the present invention.By considering explanation of the present invention disclosed herein and practice, other embodiment of the present invention for those skilled in the art will be apparent.Expect that these explanations and embodiment are only regarded as exemplary, and true scope of the present invention and spirit are indicated by appended claims.

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Claims

1. prepare density and be greater than 0.6g/cm for one kind ³the method of chitosan composite, it sequentially comprises:

A) provide the acid solution of water and chitosan;

B) neutralize described solution to form polymeric gel; And

C) described gel is dewatered simultaneously and compressed.

2. the method for claim 1, it is included in step (c) afterwards in addition, make described gel rehydrated under one or more compounds exist in water or aqueous buffer solution, described compound is selected from cross-linking agent, medicine, biological agent, nucleic acid, vaccine, immunoeffectors or its salt.

3. the method for claim 1, wherein said compositions comprises thin film or the film that thickness is less than 2mm.

4. the method for claim 1, wherein said solution comprises water, acetic acid and chitosan.

5. the method for claim 1, wherein said chitosan is be chitosan alkali form or be salt form, and described salt is selected from chitosan acetate, chitosan succinate, chitosan adipate, Sea Cure CL 313, chitosan glutamate salt, chitosan lactate, chitosan aspartate, chitosan pyruvate, chitosan phosphate, chitosan glycollate, chitosan Ascorbate, chitosan Salicylate, chitosan formates and chitosan malate.

6. the method for claim 1, wherein said acid solution comprises and is selected from following acid: formic acid, acetic acid, glycolic, citric acid, lactic acid, hydrochloric acid, glutamic acid, aspartic acid, ascorbic acid, acetone acid, malic acid, maleic acid, fumaric acid, glucuronic acid, sorbic acid, folic acid and composition thereof.

7. the method for claim 1, wherein said neutralization procedure (b) comprises makes described solution contact with hydroxide salt, and described hydroxide salt is selected from sodium hydroxide, potassium hydroxide, calcium hydroxide and magnesium hydroxide.

8. the method for claim 1, it is included in neutralization procedure (b) before in addition, to described acid solution, carries out freezing.

9. the method for claim 1, it is included in dehydration (c) before in addition, in water or aqueous buffer solution, described gel is washed.

10. the method for claim 1, it is included in dehydration (c) before in addition, described gel detergent is reached to 5.5 to 7.5 pH in water or aqueous buffer solution.

11. the method for claim 1, it is included in dehydration (c) before in addition, by the glycerite of described gel immersion 1% to 50%.

12. the method for claim 1, wherein said dehydration is included between compression period described gel is applied to vacuum.

13. the method for claim 1, it is included in described dehydration (c) before in addition, and described chitosan gel rubber is contacted with the film of selectively penetrating aqueous solution.

14. the method for claim 1 wherein, at the temperature of 2 ℃ to 150 ℃, make described chitosan gel rubber dehydration under heat exists.

15. the method for claim 1, wherein said compression step (c) is included in the minimum line pressure that applies 25 inches of Hg on described chitosan gel rubber.

16. the method for claim 1, wherein said chitosan film or film have 5.5 to 7.5 pH.

17. 1 kinds of Therapeutic Method, it comprises:

A) provide density to be greater than 0.6g/cm ³chitosan composite; And

B) described compositions is positioned on animal body or in its body.

18. methods as claimed in claim 17, wherein said chitosan composite has the 0.8g/cm of being greater than ³density.

19. methods as claimed in claim 17, wherein in described placement step (b) before, make the hydration in water or aqueous buffer solution of described compositions.

20. methods as claimed in claim 17, wherein said animal is selected from mammal and people.

21. methods as claimed in claim 17, wherein in described placement step (b) before, make described compositions in water or aqueous buffer solution, hydration under one or more compounds exist, described compound is selected from medicine, biological agent, nucleic acid, vaccine, immunoeffectors or its salt.

22. 1 kinds of compositionss, it comprises density and is greater than 0.6g/cm ³the chitosan that is thin film or form membrane.

23. compositionss as claimed in claim 22, it has 0.6g/cm ³to 1.6g/cm ³density.

24. compositionss as claimed in claim 22, it has 0.8g/cm ³to 1.6g/cm ³density.

25. compositionss as claimed in claim 22, it has 5.0 to 9.5 pH.

26. compositionss as claimed in claim 22, it contains glycerol.