KR20100137146A - Photoreactive chitosan derivatives and their uses - Google Patents

Abstract

PURPOSE: A photoreactive chitosan derivative is provided to secure safety when applied to the human body and to ensure excellent efficiency for immobilization and release of drug. CONSTITUTION: A chitosan derivative with a photoreactivity hardened by ultraviolet irradiation is represented by chemical formula 1. In chemical formula 1, R1 is a compound including an azido group, and R2 is a compound including a hydroxy group. A biocompatible tissue adhesive composition includes the chitosan derivative and shows photoreactivity hardened by ultraviolet irradiation.

Description

Photoreactive Chitosan Derivatives and Their Uses

The present invention relates to photoreactive chitosan derivatives, biotissue adhesives and substance immobilizing agents comprising the same.

Although there are many purposes for researching compounds that exhibit photoreactivity, many photosensitive polymers have been researched and developed due to the wide variety of organic polymer materials, easy molecular design, and easy molding and processing. Many applications are currently being actively conducted since they can be applied.

Particularly, polymer compounds exhibiting photoreactivity include a photosensitive portion of Cinnamic acid, a photosensitive component of azido compound, and a photosensitive portion of Naphthoquinone diazide. There is.

On the other hand, chitosan is a polysaccharide having a cation of β-1,4 bonded to glucose amine (Glucose amine) and N-acetyl glucose amine (N-Acetylglucose amine), the molecular bond is strong and hard. Chitosan is prepared by deacetylation of chitin. Such chitin is contained in the cell wall of microorganisms such as sea crustaceans such as crabs, shrimps, spiders, ants and grasshoppers, and green algae and yeasts. In particular, 25-35% of the shells of crabs and shrimps consist of chitin. These chitins occupy a particularly large proportion of the biocompatible materials present on the ground.

Chitosan made from these chitins has some characteristics of chitin. The most problematic of these is that chitosan is not dissolved in water but only in acid. If chitosan is insoluble in water, the range of application is limited and cannot be widely used. However, low molecular weight chitosan is a chemical reaction that changes the structure of the molecule and shows the property of dissolving in water, so it can be applied to various fields.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made diligent research efforts to develop a material which exhibits photoreactivity cured by ultraviolet light (UV) and can be used as a biocompatible bioadhesive and drug-fixing agent. As a result, the chitosan derivatives prepared by introducing azido groups into chitosan, preferably low molecular weight water-soluble chitosan, exhibit photoreactivity and exhibit excellent effects in bioadhesion as well as function as drug carriers by immobilizing drugs such as proteins. By confirming, this invention was completed.

Accordingly, an object of the present invention is to provide a water-soluble chitosan derivative exhibiting photoreactivity cured by ultraviolet irradiation.

Another object of the present invention is to provide a biocompatible biotissue adhesive composition comprising the above-described low molecular weight chitosan derivative and exhibiting photoreactivity cured by ultraviolet irradiation.

Still another object of the present invention is to provide a substance fixative comprising the chitosan derivative described above.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a chitosan derivative of formula 1 exhibiting photoreactivity cured by ultraviolet irradiation:

Formula 1

In the above formula, R ₁ is a compound containing hydrogen, an azido group or an azido group; R ₂ is a compound containing a hydroxyl group; At least one of R ₁ and R ₂ is a compound comprising an azido group or an azido group; When R ₂ is a hydroxyl group, it may be substituted or unsubstituted.

The present inventors have made diligent research efforts to develop a material which exhibits photoreactivity cured by ultraviolet light (UV) and can be used as a biocompatible bioadhesive and drug-fixing agent. As a result, chitosan derivatives prepared by introducing azido groups into chitosan, preferably low molecular weight water-soluble chitosan, exhibit photoreactivity and exhibit excellent effects in bioadhesion as well as act as drug carriers by immobilizing drugs such as proteins. It was confirmed.

The photoreactive chitosan derivative of the present invention is represented by the formula (1). That is, the chitosan derivative of the present invention includes all polymers in which azido groups are introduced into chitosan or a derivative thereof.

In the general formula (1), the term “compound comprising azido group” means a radical derived from an aliphatic compound or aromatic compound containing azido group. For example, the compound containing an azido group may be azidobenzoic acid, 4,4'-diazidostilbene, 4,4'-diazidochalcone, 4,4'-diazidobenzophenone, 2,6-bis ( 4'-azidobenzylidene) cyclohexanone, 1-azidopyrene, 1,6-diazidopyrene, 2- (4'-methoxyanilino) -5-azidobenzoic methyl ester, 2 -Anilino-5-azidobenzoic acid, 2- (4'-azidophenyl) -6-methyl benzothiazole or 2- (4'-azidophenyl)-(naphthol-1 ', 2'-4, Radicals derived from 5-oxazole).

In Formula 1, when R ₂ is a hydroxyl group, it may be substituted, for example, may be substituted with a carboxyl group.

According to a preferred embodiment of the invention, R ₁ in formula ₁ is a compound comprising an azido group; R ₂ is a compound containing an unsubstituted or substituted hydroxyl group, azido group or azido group.

More preferably, the chitosan derivative of the present invention is a photoreactive chitosan derivative represented by the following formula (2):

Formula 2

In the above formula, R ₂ is a compound containing an unsubstituted or substituted hydroxyl group, azido group or azido group; n is an integer of 2-100,000.

Even more preferably, the chitosan derivative of the present invention is a chitosan derivative represented by the following formula (3), (4) or (5):

Formula 3

In the above formula, n is an integer of 2-100,000.

Formula 4

In the above formula, n is an integer of 2-100,000.

Formula 5

In the above formula, n is an integer of 2-100,000.

Most preferably, the chitosan derivative of the present invention is a chitosan derivative represented by the formula (3) or (5).

According to another preferred variant of the invention, the chitosan derivative of the invention is a chitosan derivative represented by the following formula (6):

6

In the above formula, R ₁ is a compound containing hydrogen, an azido group or an azido group; n is an integer of 2-100,000.

More preferably, the chitosan derivative of the present invention is a chitosan derivative represented by the following formula (7):

Formula 7

In the above formula, n is an integer of 2-100,000.

According to a preferred embodiment of the present invention, the chitosan used in the present invention is a low molecular weight chitosan. As used herein, the term “low molecular weight chitosan” refers to chitosan with an average molecular weight of 200-100,000, preferably with an average molecular weight of 500-50,000, more preferably with an average molecular weight of 1,000-50,000, and most preferably with an average molecular weight of 1,000-30,000. Chitosan is divided into oligomers of about 12 monomers and polymers belonging to a polymer according to the number of monomers connected, and the polymer is a low molecular weight chitosan having a molecular weight of less than 150,000, a polymer chitosan having a molecular weight of 700,000 to a million, and the space between It is divided into the middle molecule chitosan having the range of.

More preferably, in the above formulas n is 10-10,000, even more preferably n is 20-1,000, even more preferably n is 20-500, even more preferably n is 20-100, most preferably n is 20-80.

According to a preferred embodiment of the present invention, the chitosan used in the present invention is a low molecular weight water soluble chitosan.

The photoreactive chitosan derivatives of the present invention exhibit safety without showing toxicity or side effects to the living body, and are hardened by photoreactions and exhibit adhesiveness, so that the bioadhesives and drug-fixing agents are used for living tissues or bioadhesive or drug fixation. Can function as

According to another aspect of the present invention, the present invention provides a biocompatible biotissue adhesive composition comprising the chitosan derivative of the present invention described above and exhibiting photoreactivity cured by ultraviolet irradiation.

Since the biocompatible biotissue adhesive composition of the present invention contains the above-mentioned low molecular weight chitosan derivative of the present invention as a main component, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

The bioadhesive and drug-fixing formulations of the present invention can replace the cyanoacrylic adhesives currently commonly used in the market. For example, the biocompatible biotissue adhesive of the present invention can replace surgical sutures, can be used to block unnecessary blood vessels, and can be used for hemostasis and suturing of soft tissues such as facial tissues, cartilage, and hard tissues such as bones and teeth. It is possible to apply as a home medicine.

Various applications of the biocompatible biotissue adhesive composition of the present invention are summarized as follows:

The biotissue adhesive of the present invention may be applied to the internal and external surfaces of the human body, that is, the biotissue adhesive of the present invention may be applied to an external surface of the human body (eg, skin) or an internal surface such as the surface of an internal organ exposed during a surgical procedure. It can be applied topically.

The biotissue adhesives of the present invention can be used to bond the mouse portion of a tissue, to seal leakage of air / fluid from the tissue, to adhere a medical device to the tissue, or to fill a defective portion of the tissue. The term “biotissue” herein is not particularly limited and includes, for example, skin, bone, nerve, axon, cartilage, blood vessels, cornea, muscle, fascia, brain, prostate, breast, endometrium, lung, spleen, small intestine. , Liver, testes, ovaries, cervix, rectum, stomach, lymph nodes, bone marrow and kidneys.

The biotissue adhesive of the present invention can be used for wound healing. For example, the biocompatible biotissue adhesive of the present invention can be used as a dressing applied to a wound.

The biotissue adhesive of the present invention can be used for skin closure. In other words, the biotissue adhesive of the present invention may be applied locally and used to seal the wound, thereby replacing the suture. In addition, the biological tissue adhesive of the present invention can be applied to hernia restoration, for example, can be used for the surface coating of the mesh used for hernia restoration.

The biotissue adhesives of the present invention can be used to prevent closure and leakage of tubular structures such as blood vessels. In addition, the biological tissue adhesive of the present invention can also be used for hemostasis.

Meanwhile, the biotissue adhesive of the present invention may be used as an anti-adhesion agent after surgery. Adhesion occurs at all surgical sites, where other tissues stick around the wound around the surgical site. Adhesion occurs 97% after surgery, especially 5-7% of which causes serious problems. To prevent these adhesions, the wound may be minimized during surgery or anti-inflammatory agents may be used. It also activates tissue plasminogen activators (TPAs) or physical barriers such as crystalline solutions, polymer solutions, and solid membranes to prevent fibrin formation, but these methods can be toxic in vivo and have other side effects. have. The biotissue adhesive of the present invention may be applied to exposed tissue after surgery to be used to prevent adhesions occurring between the tissue and surrounding tissue.

According to a preferred embodiment of the present invention, the chitosan derivative used as the biotissue adhesive of the present invention is an ultraviolet exposed chitosan derivative.

According to another aspect of the present invention, the present invention provides a substance fixative comprising the chitosan derivative of the present invention described above.

Since the substance stabilizer of the present invention contains the above-described low molecular weight chitosan derivative of the present invention as a main component, the contents common between the two are omitted in order to avoid excessive complexity of the present specification.

The substance fixative of the present invention may be expressed as a "drug immobilizer" or a "drug delivery system."

Substances which can be immobilized by the drug fixing agent of the present invention are not particularly limited and include chemicals, small molecules, polypeptides (proteins), peptides, nucleic acids or viruses.

Small molecules to be immobilized in the present invention may be, for example, drugs, contrast agents (e.g., T1 contrast agent, T2 contrast agent such as superparamagnetic material, radioisotopes, etc.), fluorescent markers, dyeing materials, and the like, but are not limited thereto. Do not. A polypeptide is a polymer of amino acids consisting of two or more residues and includes peptides and proteins. Polypeptides include, for example, proteins involved in cell immortality (eg, SV40 large T antigen and telomerase), anti-apoptotic proteins (eg, mutant p53 and BclxL), antibodies, oncogenes (eg, ras, myc, HPV). E6 / E7 and adenovirus Ela), cell cycle regulatory proteins (eg cyclin and cyclin dependent kinase) or enzymes (eg green fluorescent protein, beta-galactosidase and chloramphenicol acetyl transferase), It is not limited to this. In addition, the nucleic acid can be, for example, RNA, DNA or cDNA, and the sequence of nucleic acids can be coding site sequences or non-coding site sequences (eg, antisense oligonucleotides or siRNAs). The virus can be a virus core (ie, a nucleic acid of a virus packaged without the envelope of the virus) comprising the virus or the nucleic acid of the virus. Examples of viruses and viral cores that can be transported include, but are not limited to, papilloma virus, adenovirus, baculovirus, retrovirus core and semilky virus core. Nucleotides as nucleic acid cargo may be standard nucleotides (eg adenosine, cytosine, guanine, thymine, inosine and uracil) or analogs (eg phosphorothioate nucleotides). For example, the nucleic acid cargo may be an antisense sequence or RNAi consisting of phosphorothioate nucleotides.

According to a preferred embodiment of the invention, the substance immobilized by the immobilizing agent of the invention is a protein.

Proteins carried by the immobilizing agent of the present invention are not particularly limited and include, but are not limited to, growth factors, hormones, hormonal analogues, enzymes, inhibitors, signaling proteins or portions thereof, antibodies or portions thereof, short chain antibodies, binding proteins or bindings thereof. Domains, antigens, adhesion proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators, blood coagulation factors and vaccines, and the like. More specifically, the protein immobilized by the immobilizing agent of the present invention is insulin, IGF-1 (insulin-like growth factor 1), growth hormone, erythropoietin, G-CSFs (granulocyte-colony stimulating factors), Granulocyte / macrophage-colony stimulating factors (GM-CSFs), interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin-3, interleukin-4, interleukin-6, interleukin-2, and epidermal growth factors ), Calcitonin (calcitonin), adrenocorticotropic hormone (ACTH), tumor necrosis factor (TNF), atobisban, buserelin, cetrorelix, deslorelin, des Depresso, dynorphin A (1-13), elcatonin, eleidosin, eptifibatide, growth hormone releasing hormone- GHRH-II II), gonadorelin, goserelin, hystrelin, leuprolerin (l euprorelin, lypressin, octreotide, oxytocin, phytocin, pitressin, secretin, sincalide, terlipressin, thymopentin ), Thymosine α1, triptorelin, bivalirudin, carbetocin, cyclosporin, exedine, lanreotide, LHRH (luteinizing hormone) include, but are not limited to, -releasing hormone, nafarelin, parathyroid hormone, pramlintide, T-20 (enfuvirtide), thymalfasin, and ziconotide.

According to a preferred embodiment of the present invention, the chitosan derivative used as the immobilizing agent of the present invention is an ultraviolet exposed chitosan derivative.

As demonstrated in the examples below, the immobilizing agents of the present invention have adhesion to cells. This property allows the use of a small amount of drug to increase the local concentration of the drug to exhibit higher activity. In addition, the immobilizing agent of the present invention is a cytotoxic and safe material, the efficiency of immobilizing the drug and the time to release can be improved by increasing the amount of chitosan derivatives.

The features and advantages of the present invention are summarized as follows:

(a) The chitosan derivative of the present invention is prepared by introducing an azido group into chitosan.

(b) The chitosan derivative of the present invention is biocompatible and has little toxicity as a biotissue adhesive, and thus can be safe when applied to human body.

(c) Chitosan derivatives of the present invention can be used as tissue adhesion, wound healing, skin closure, suture and leakage prevention of vascular structures such as blood vessels, hemostasis or anti-adhesion.

(d) The chitosan derivatives of the present invention exhibit excellent efficiency in terms of immobilization and release of drugs, and have high potential for application as drug carriers because they are not toxic.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experimental material

Chitosan was purchased from Jawang (Korea) and the degree of deacetylation (DAc) was 88%. Chitosan was used without further purification. Acetic acid, sodium nitride, ammonium hydroxide, sodium tetrahydridoborate, methanol, acetone and ether used to obtain low molecular weight chitosan were purchased from Deoksan Pharmaceutical Co., Ltd. N-hydroxysuccinimide, N, N-dicyclohexylcarbodiimide and diethyl ether for preparing N- (4-azidobenzoyloxy) succinamide are prepared from Wako Pure Chem. Purchased from Ind. (Japan), 4-azidobenzoic acid was obtained from Tokyo Kasei Kogyo Co., Ltd. (Japan) and dioxane were purchased from Samjeon Chemical (Korea). 2,2-azobis (isobutyronitrile) is available from Wako Pure Chem. It was purchased from Ind. (Japan). Poly (ethylene glycol) methacrylate and 4-azidophenyl methacrylate were purchased from Sigma-Aldrich (USA) and used to prepare azido-poly (ethylene glycol) (Az-PEG), in which case Acta biomaterialia Inc. followed the method of Yoshihiro Ito et al. [22]. For cell culture, protein release testing and MTT analysis, Dulbecco's Modified Eagle's Medium (DMEM) was purchased from WelGene Inc., and fetal bovine serum (FBS) and penicillin-streptomycin were purchased from GIBCO. Trypsin-EDTA, (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, terrazole) MTT formazan and Bovine Serum Albumin (BSA) were obtained from Sigma-Aldrich. Purchased. Cell culture coverslips, Sterile, Thermanox plastic (15 mm diameter) were purchased from NUNC (United States). 6 well and 96 well plates were purchased from Becton Dickinson Labware (USA) and TPP Immunomaxi (Switzerland), respectively. African Green Monkey SV40-transf'd kidney fibroblast cell line (COS7) cells, an African green monkey kidney cell derived from CV-1, Cercopithecus aethops , were purchased from Riken Cell Bank. 3T3 (mouse embryonic fibroblast cell line) cells were obtained from Kyungpook National University School of Medicine.

Example 1 Preparation of Low Molecular Weight Water Soluble Chitosan

25 g of chitosan Co., Ltd. was dissolved in an aqueous solution of 4% (v / v) acetic acid, and then an aqueous sodium nitrite solution (4.35 g of NaNO ₂ dissolved in 43.47 ml of distilled water) was dissolved in free amine of chitosan while stirring in an ice bath. 0.5 molar ratio per minute. After the reaction for 3 hours, the mixture was neutralized to pH 7.4 with ammonia water. 10.55 g of NaBH ₄ in a 2 molar ratio was added to the prepared low molecular weight aqueous chitosan solution in small amounts as it was, while reducing the terminal group generated during the deamination reaction, and neutralizing to pH 7.4 with 1 N aqueous HCl solution. Chitosan oligomers obtained by nitrous acid decomposition were filtered to remove insoluble materials, and water, methanol, and acetone were fractionated and precipitated using a mixed solution having different mixing ratios. The low molecular weight chitosan used in this study was fractionated and precipitated from a mixed solution of water and methanol in a ratio of 1: 3. After filtration, the mixture was washed with acetone and ether and dried in vacuo to obtain a powdery chitosan oligomer. Azidophenyl chitosan derivatives were prepared using chitosan oligomers having an average molecular weight of about 10,000 in the chitosan.

Meanwhile, the molecular weight of low molecular weight chitosan was determined using HPLC (high performance liquid chromatography, Gilson, France) with pullulan standard (column, Shodex Asahipak GS-320, 30 cm X 7.6). The mobile phase consists of 0.2 M CH ₃ COOH and 0.036 M CH ₃ COONa. Samples of 0.1% (w / v) were loaded and eluted at a flow rate of 0.5 ml / min at 25 ° C. The eluted peak was detected by RI detector (132 RI detector, Gilson, France). Major macrostructures and DAc of low molecular weight chitosan were identified by Fourier-transform infrared (FT-IR) and proton nuclear magnetic resonance ( ¹ H-NMR) analysis, respectively. FT-IR spectra were obtained using KBr pellets on an FT-IR spectrophotometer (FT-IR 8400s, Shimadzu, Japan). ¹ HNMR spectra were obtained using D ₂ O and CD ₃ COOD as solvent and NMR spectrophotometer (Gemini 2000, 300 MHz, Varian, USA). 10 mg samples were placed in a 5 mm NMR test tube with 0.5 ml D2O / CD3COOD solution.

As a result of the FT-IR spectrum, the IR spectrum of the low molecular weight chitosan was similar to that of the high molecular weight chitosan polymer. This shows that the main macromolecular structure of the chitosan polymer remains intact in the low molecular weight chitosan.

The degree of deacetylation (DAc) of the chitosan polymer and the low molecular weight chitosan was determined by ¹ H NMR spectroscopy. The DAc of chitosan polymers and low molecular weight chitosans is the intensity of integration of CH ₃ residues on acetamide groups (I _acetyl-H ) and the intensity of integration of H ₂ , H ₃ , H ₄ , H ₅ , H ₆ and H _{6 ′} protons. It was evaluated by the sum of (I _acetyl-H ) [24].

DAc (%) = [1-((1/3) I _{acetyl H} / (1/6) I _H2-H6 )] x 100

The resonance at 2.04 ppm was due to the CH ₃ residue on the acetamide group, and the resonance of H ₂ -H ₆ occurred in the range of 3.1-4.0 ppm [25]. The DAc values of the chitosan polymer and the low molecular weight chitosan were 87.62% and 86.24%, respectively. The weight average molecular weight (Mw) of the low molecular weight chitosan was determined by HPLC analysis and measured to be about 10,973.

Example 2 Preparation of Azido Phenylated Low Molecule Water Soluble Chitosan

2 g of low molecular weight chitosan having an average molecular weight of about 10,000 obtained above was dissolved in 30 ml of water. To this aqueous solution was added 1.15 g of 4-azidobenzoyloxysuccinimide dissolved in 20 ml of dioxane under a cold bath. After reacting at 60 ° C. for 3 hours, the solvent was removed under reduced pressure, and then sufficiently washed with acetone and dried to prepare an azidophenyl-chitosan derivative of Chemical Formula 3. The major macrostructures of azidophenyl chitosan derivatives were analyzed by Fourier-transform infrared (FT-IR) and proton nuclear magnetic resonance ( ¹ H-NMR) analysis. According to the FT-IR spectrum (FIG. 1), the spectral profile of the azidophenyl-chitosan derivative was similar to that of low molecular weight chitosan and chitosan polymers. This result indicates that the azidophenyl-chitosan derivative maintains the pranose structure of the low molecular weight chitosan and chitosan polymer. The IR spectrum of the azidophenyl-chitosan derivative shows a characteristic absorption band near 2125 cm ⁻¹ , indicating the presence of azido groups. In order to verify the structure of the azidophenyl chitosan derivative, ¹ H-NMR spectroscopy analysis was performed (FIG. 2). Aromatic rings were analyzed at 7.0-8.0 ppm in the ¹ H-NMR spectrum, indicating a benzene ring as part of the azidophenyl group. Thus, it can be seen that azidophenyl chitosan was produced successfully.

Azidophenyl groups were introduced into the amino groups of low molecular weight chitosan during the preparation of azidophenyl chitosan derivatives. The degree of introduction of the azidophenyl group was determined by ¹ H-NMR spectrum. The DAc formula of the low molecular weight chitosan described above was used and the degree of introduction of azidophenyl groups was determined according to the following equation: D (%) = (1/4 XI _benzeneH ) / (1/6 XI _H2-H6 ) X 100

Example 3: Photoreactivity Degree with Time of 1.0 wt% Azidophenyl-chitosan Derivative

0.1 g of the azidophenyl-chitosan derivative prepared in Example was dissolved in 9.9 g of water to prepare an aqueous solution at 1.0% by weight. 20 µl of this aqueous solution was added dropwise onto the glass plate, followed by ultraviolet (UV) irradiation, and the glass plate was placed at a right angle to investigate photoreactivity with time. The light irradiator used to irradiate ultraviolet rays used an SP-9 spot cure system (USHIO, Inc.). The control group did not irradiate light and compared with the experimental group was set to show the difference in the degree of curing according to the irradiation time. The difference was depending on the time of light irradiation. The experimental results are shown in Table 1 and FIG. The photocuring degree was calculated by the following method: Curing degree = (flowing distance / flowing distance when no light was irradiated)

The light irradiation results of the experimental group and the control group were washed in running water for 10 minutes. At this time, the photoreactivity exhibited a function as a bioadhesive was not washed off under running water, and the longer the irradiation time was, the better the adhesion to the coverslip was.

Photoreactivity degree with time of 1.0 wt% azidophenyl-chitosan derivative Light irradiation time
(sec) Light curing degree
(%) 0 0 30 8 60 13 90 18 120 15 150 19 180 26 210 34 240 100 270 100 300 100

As can be seen in Table 1 and Figure 3, the azidophenyl-chitosan derivatives of the present invention exhibited excellent photoreactivity, so that the degree of light curing increased with the passage of UV irradiation time.

Example 4: Photoreactivity Degree with Time of 3.0 wt% Azidophenyl-chitosan Derivatives

The experiment was carried out in the same manner as in Example 3, but the concentration of the azidophenyl-chitosan derivative was adjusted to 3.0% by weight. The experimental results are shown in Table 2 and FIG.

Degree of photoreactivity with 3.0 wt% azidophenyl-chitosan derivatives over time Light irradiation time
(sec) Light curing degree
(%) 0 0 30 15 60 12 90 26 120 38 150 100 180 100 210 100

As can be seen in Table 2 and Figure 4, the 3.0 wt% azidophenyl-chitosan derivative of the present invention exhibited a pattern that cured in less time than the 1.0 wt% azidophenyl-chitosan derivative. For example, 1.0% by weight azidophenyl-chitosan was 240 seconds while the time to show 100% light cure was 3.0 seconds by weight, while 3.0% by weight azidophenyl-chitosan was 150 seconds.

Example 5: Adhesion Analysis Using Azidophenyl-chitosan Derivatives

In order to test the adhesion of azidophenyl-chitosan to the connective tissues of animals, the adhesion of azidophenyl-chitosan derivatives was investigated using so-called pig skins with commercially available porcine skin tissue. Pork rinds were cut into (width × length × height) (5 cm × 5 cm × 1.5 cm). 10 wt% azidophenyl-chitosan derivative was applied to the cleavage site, and then UV was irradiated for 5 minutes to allow the cleavage site to adhere. As can be seen in Figure 5, the azidophenyl-chitosan derivative exhibited the photoreactivity to act as a suitable adhesive for the living body showed the effect of re-adhesion of the cut out.

Example 6: Difference in Curing Degree of Azidophenyl-chitosan Derivatives with or Without UV Irradiation

Prior to the experiment, we compared the ^TM Thermanox plastic cover's ripsang and Az-PEG in the COS7 cell culture (azido-polyethylene glycol) Thermanox plastic cover ^TM's ripsang which is coated on the surface. As can be seen in Figures 6 and 7, the actual cells are well cultured on the Thermanox ^TM plastic coverslip, but almost no cell culture when Az-PEG is coated thereon.

The azidophenyl-chitosan material was recoated on Az-PEG having such conditions that cells were not well cultured. Az-PEG prevents the cultured cells from adhering to the coverslip. Therefore, if azidophenyl-chitosan is not cured by UV, a large amount of water will be washed away by washing with running water. As a result, exposed parts coated with Az-PEG will not adhere and proliferate. On the other hand, if the azidophenyl-chitosan is cured by UV, most of the azure phenyl-chitosan will lose its water solubility and will not be exposed even if it is washed with running water. As a result, in this case, the cells will be able to attach and proliferate. Based on this hypothesis, the cells were incubated on the cover slip coated with Az-PEG and 0.1% by weight of azidophenyl-chitosan, irradiated with UV on sufficiently grown cells, and placed on flowing water for 30 minutes so that the non-adhesive cells were separated. It was. As a result of the experiment, UV-irradiated cells adhered well to the coverslip by the adhesion of azidophenyl-chitosan by photoreactivity, whereas cells washed in flowing water without UV irradiation for 30 minutes did not show adhesion. It was washed out in water (see FIGS. 8 and 9). Thus, it can be seen that the cells bind strongly to azidophenyl-chitosan derivatives.

The growth factor concentration is a very important factor in increasing the reaction rate between the derivative of the cell and the growth factor or increasing the activity of the growth factor. A simple way to increase the concentration of growth factors is to use higher growth factors. However, the use of a large amount of growth factors are commercially problematic and have a high probability of causing side effects. Reducing the growth factor dose can reduce side effects [7]. If immobilized growth factors are released to derivatives on the cell surface, the local concentration of growth factors around the cell derivatives increases. In this case, a smaller growth factor is needed. According to the experimental results, azidophenyl-chitosan derivatives immobilized with growth factors can bind to the cell surface. Thus, azidophenyl-chitosan derivatives can reduce the amount of growth factor required and can also exhibit higher growth factor activity compared to free growth factors. The azidophenyl-chitosan derivatives of the present invention increase the local concentration of growth factors on the cell surface so that they can exhibit higher growth factor activity [26].

Example 7: Cytotoxicity Test of Azidophenyl-chitosan Derivatives

In order to test the cytotoxicity of UV exposed azidophenyl chitosan derivatives, it is not suitable to use general MTT assays, since azidophenyl chitosan derivatives exposed to UV are water insoluble. Samples of various concentrations (5 w / v%, 10 w / v%, 15 w / v% and 20 w / v%) of azidophenyl chitosan derivative solutions were prepared. The samples were then exposed to UV for various times (1 minute, 3 minutes, 5 minutes and 7 minutes) to coat the surface of the wells in 6 well culture plates. 3T3 (mouse embryonic fibroblast line) was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum and 1% penicillin-streptomycin at 37 ° C and 5% CO ₂ /95% atmospheric air. . The cells were seeded at a density of 1.0 × 10 ⁴ cells / well in 6 well culture plates coated with azidophenyl chitosan derivatives. The medium was then replaced with 200 μl of a suitable test medium (serum-free DMEM) and the plates were incubated for 48 hours at 37 ° C., 5% CO ₂ /95% atmospheric air. Cells were also cultured under the same conditions as above in a plate (control) not coated with azidophenyl chitosan derivative. MTT formazan was added to the plate and allowed to react for 4 hours. DMSO was added to each plate and the mixture in the plate was transferred to a 96 well plate. Then, a conventional MTT assay method was performed.

In order to apply azidophenyl chitosan derivatives to growth factors used in medicine, it is necessary to confirm whether azidophenyl chitosan derivatives are cytotoxic. As a result of the MTT assay, all azidophenyl chitosan derivative samples were not cytotoxic and did not affect the proliferation of 3T3 cells at 48 hours after treatment of azidophenyl chitosan derivative samples in serum-free DMEM.

Example 8: Surface observation of azidophenyl-chitosan derivatives

The mixture of azidophenyl chitosan derivative and BSA solution was exposed to UV. Subsequently, the surfaces of the azidophenyl chitosan derivatives and the BSA conjugates were observed with a runner electron microscope (S-3400N, Hitachi, Japan).

SEM (Scanning Electron Microscope) results of the azidophenyl chitosan derivative and the azidophenyl chitosan derivative-BSA are shown in FIG. 10. The azidophenyl chitosan derivatives dissolved in water were converted to macromolecules by binding to each other when exposed to UV. BSA was coated or surrounded by UV exposed azidophenyl chitosan derivatives. It is expected that high activity will be shown by the inhibition of incorporation [26].

Example 9: Protein Release Assay

Various concentrations (5%, 10%, 15%, 20%, w / v) of azidophenyl chitosan derivative solution samples were prepared (4 samples per same concentration, 16 samples in total, 4 experimental groups for one experimental group). . 200 μg of BSA was dissolved in each sample solution. Each sample experimental group was exposed to UV for 1 minute, 3 minutes, 5 minutes and 7 minutes. All samples were washed three times with PBS (phosphate buffer saline) to remove BSA that was not immobilized with azidophenyl chitosan derivatives and 1 ml of PBS was added to each sample. The amount of released BSA was analyzed every 12 hours according to the Bradford assay (Bio-rad protein assay, Bio Rad laboratories, Inc.) and UV / VIS spectrophotometer (Optizen 3220UV, Mecasys Co., Ltd. Korea) . Samples of the aforementioned concentrations without BSA were used as controls. After all measurements, 1 ml of PBS was added to each sample.

In order to determine the optimal UV exposure time and azidophenyl chitosan derivative concentration for the highest growth factor immobilization efficiency of the azidophenyl chitosan derivative, a protein release experiment was conducted. Samples were divided into four experimental groups by concentration. Samples of 20 w / v% or more could not be prepared due to the solubility of the azidophenyl chitosan derivative. The saturation point of the azidophenyl chitosan derivative is determined to be 20 w / v%. The amount of BSA released decreased over time. A small amount of BSA was released early in the experiment and there was a significant change in the amount of BSA released. However, after 36 hours, the amount of BSA released was significantly reduced and there was little change in the amount of BSA released. According to the experimental results, the correlation between the amount of immobilized BSA and the concentration of azidophenyl chitosan derivative used for immobilization is clearly shown. The average amount of BSA released from each experimental group was about 36.6 μg in A test group, 93.9 5 μg in B test group, 134.49 μg in C test group, and 162.96 μg in D test group. Since the amount of BSA released can be regarded as the amount of BSA immobilized by the azidophenyl chitosan derivative, the BSA 18.3% in A experimental group, 46.98% in B experimental group, 67.25% in C experimental group compared to the amount of BSA used for initial immobilization. And 81.48% in D experimental group was determined to be immobilized by the azidophenyl chitosan derivative. According to the experimental results, it can be seen that the amount of immobilized BSA increases in proportion to the concentration of azidophenyl chitosan derivative used for immobilization. However, there was no clear correlation between UV exposure time and immobilized BSA amount of azidophenyl chitosan derivatives. BSA of each experimental group was released with 276 hours in A experimental group, 336 hours in B experimental group, 420 hours in C experimental group and 432 hours in D experimental group. According to the experimental results, it can be seen that the higher the concentration of the azidophenyl chitosan derivative, the longer the BSA release period. Therefore, when the concentration of the azidophenyl chitosan derivative used for immobilization is the highest in the range below the saturation concentration, the highest efficiency can be obtained in terms of immobilization amount and release time of the growth factor.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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1 is an FT-IR spectrum of low molecular weight chitosan (a) and azidophenyl-chitosan derivatives (b).

Figure 2 is a 300 MHz ¹ H-NMR spectrum of low molecular weight chitosan (a) and azidophenyl-chitosan derivative (b) in D2O in the presence of CD3COOD.

Figure 3 is a graph showing the degree of photoreactivity with time of the 1.0 wt% azidophenyl-chitosan derivative of the present invention.

Figure 4 is a graph showing the degree of photoreactivity with time of the 3.0 wt% azidophenyl-chitosan derivative of the present invention.

Figure 5 is a photograph showing the re-adhesion of the cut pig skin tissue by the azidophenyl-chitosan derivative of the present invention.

6 is a photograph showing the cells on the coverslip surface after culturing and washing the cells on the plastic coverslip.

7 is a photograph showing the cells on the coverslip surface after culturing and washing the cells on a plastic coverslip coated with AZ-PEG (azido-polyethylene glycol). No cells are observed on the coverslip surface.

8 is a photograph showing the cells on the coverslip surface after culturing the cells on a plastic coverslip coated with azidophenyl-chitosan, irradiated with UV (ultraviolet) and then washed. The adhesion of azidophenyl-chitosan by photoreactivity can be observed to adhere well to the coverslip.

Figure 9 is a photograph showing the cells on the coverslip surface after culturing and washing the cells on a plastic coverslip coated with azidophenyl-chitosan. In the absence of UV irradiation, the cells did not adhere to the surfaces of the coverslips and were washed out with water.

10A-10C are scanning electron microscope (SEM) analysis images of azidophenyl-chitosan derivatives (FIG. 10A) and azidophenyl-chitosan derivatives-BSA (FIG. 10B, FIG. 10C) exposed to UV.

Claims (11)

Chitosan derivatives of the general formula (1) exhibiting photoreactivity cured by ultraviolet irradiation: Formula 1

In the above formula, R ₁ is a compound containing an azido group; R ₂ is a compound containing a hydroxyl group. The compound of claim 1, wherein R ₁ is a compound including an azido group; R ₂ is a chitosan derivative, characterized in that the compound containing an unsubstituted or substituted hydroxyl group. The compound of claim 2, wherein R ₁ is a compound including an aryl azido group; R ₂ is a chitosan derivative, characterized in that the compound containing an unsubstituted or substituted hydroxyl group. The chitosan derivative according to claim 3, wherein the chitosan derivative is represented by the following Chemical Formula 2: Formula 2

In the above formula, R ₂ is a compound containing an unsubstituted or substituted hydroxyl group, azido group or azido group; n is an integer of 2-100,000. The chitosan derivative according to claim 4, wherein the chitosan derivative is represented by the following Chemical Formula 3: Formula 3

In the above formula, n is an integer of 2-100,000. The chitosan derivative according to any one of claims 1 to 5, wherein n is an integer of 20-1,000. A biocompatible biotissue adhesive composition comprising the chitosan derivative of any one of claims 1 to 5 and exhibiting photoreactivity cured by ultraviolet irradiation. 8. The biocompatible biotissue adhesive composition according to claim 7, wherein n is an integer of 20-1,000. A substance fixative comprising the chitosan derivative of any one of claims 1 to 5. 10. The substance fixative of claim 9 wherein n is an integer from 20 to 1,000. 10. The substance fixative of claim 9, wherein the substance is a protein.

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