Modulation of macrophage polarity with carboxymethyl chitin gated hollow mesoporous silica nanoparticles for elevating anti-tumor chemotherapy.

The weak immunity of tumors after chemotherapy could cause tumor metastasis and progression. Therefore, to overcome the dilemma of obvious immune deficiency caused by chemotherapy, a nanosystem (N-IL-12/DOX/α-TOS) consisted of thioketal (TK) bonds linked-hollow mesoporous silica nanoparticles (HMSNs) coated with carboxymethyl chitin (CMCH) by electrostatic interaction, and surface-functionalized glucose-regulated protein 78 binding peptide was prepared for loading doxorubicin (DOX), IL-12 and α-tocopheryl succinate (α-TOS). N-IL-12/DOX/α-TOS displayed a mean size of 275 nm after encapsulated DOX, IL-12 and α-TOS with loading contents of 2.04 × 10-4, 4.01 × 10-2 and 7.12 × 10-2, respectively. The drug-free nanoparticles (NPs) showed good biocompatibility to both 4 T1 cells and RAW264.7 macrophages. N-IL-12/DOX/α-TOS could achieve localized release of IL-12, DOX and α-TOS by pH and H2O2 trigger in the tumor microenvironment (TME). Moreover, the combined therapy by N-IL-12/DOX/α-TOS remarkably elevated the anti-tumor therapeutic efficacy, enhanced immune responses via promoting tumor-associated macrophage (TAM) polarization into tumoricidal M1 phenotypes, and decreased lung metastasis with reduced side effects. N-IL-12/DOX/α-TOS exhibited as a promising strategy for combining chemotherapy and local macrophage modulation-immunotherapy for anti-tumor therapy.

Hemostasis-osteogenesis integrated Janus carboxymethyl chitin/hydroxyapatite porous membrane for bone defect repair.

Barrier membranes with osteogenesis are desirable for promoting bone repair. Janus membrane, which has a bilayered structure with different properties on each side, could meet the osteogenesis/barrier dual functions of guided bone regeneration. In this work, new biodegradable Janus carboxymethyl chitin membrane with asymmetric pore structure was prepared based on thermosensitive carboxymethyl chitin without using any crosslinkers. Nano-hydroxyapatites were cast on single-sided membrane. The obtained carboxymethyl chitin/nano-hydroxyapatite Janus membrane showed dual biofunctions: the dense layer of the Janus membrane could act as a barrier to prevent connective tissue cells from invading the bone defects, while the porous layer (with pore size 100-200 µm) containing nano-hydroxyapatite could guide bone regeneration. After implanted on the rat critical-sized calvarial defect 8 weeks, carboxymethyl chitin/nano-hydroxyapatite membrane showed the most newly formed bone tissue with the highest bone volume/total volume ratio (10.03 ± 1.81 %, analyzed by micro CT), which was significantly better than the commercial collagen membrane GTR® (5.05 ± 0.76 %). Meanwhile, this Janus membrane possessed good hemostatic ability. These results suggest a facile strategy to construct hemostasis-osteogenesis integrated Janus carboxymethyl chitin/hydroxyapatite membrane for guided bone regeneration.

Biodegradable carboxymethyl chitin-based hemostatic sponges with high strength and shape memory for non-compressible hemorrhage.

Uncontrolled hemorrhage of deep, narrow and non-compressible perforating wounds is responsible for many trauma deaths. In this work, a novel biodegradable hemostatic sponge based on thermosensitive carboxymethyl chitin was prepared via simple cryo-regeneration process without using any crosslinkers. The collagen and polydopamine were added to further enhance mechanical and hemostatic properties of the sponge. All the carboxymethyl chitin based sponges showed high strength with excellent water/blood-triggered shape memory property, and the highest compressive fracture wet-strength could reach about 291.2 kPa, which was almost higher than those of many reported biodegradable hemostatic sponges pre-swelled in water. More importantly, the carboxymethyl chitin-collagen-polydopamine sponges displayed much better blood-clotting capacity and superior hemostasis performance than gauze and clinically used collagen sponge iRegene@ in vitro and in the rat liver perforating wound model. This study revealed a facile strategy to construct the effective carboxymethyl chitin based hemostatic sponges for the deep and non-compressible perforating wound.

Size-tunable and biodegradable thrombin-functionalized carboxymethyl chitin microspheres for endovascular embolization.

As a minimally invasive method, endovascular embolization has been an effective strategy for controlling bleeding and tumor treatment. Herein, carboxymethyl chitin embolic microspheres were prepared with the aqueous two-phase carboxymethyl chitin/polyethylene glycol system without using any crosslinking agents and thrombin-functionalized embolic microsphere named as Thr@CMCHm-30 was made after covalent introduction of thrombin. The size of the microspheres can be adjusted from 5 to 500 µm. The data of in vitro and in vivo tests indicated that these microspheres possessed good degradability and biocompatibility. Meanwhile, Thr@CMCHm-30 can significantly promote blood clotting and enhance the strength of the blood clots. More importantly, Thr@CMCHm-30 displayed better embolization effect than that of the commercial available Gelfoam Alicon® and polyvinyl alcohol-based embolic microspheres CalliSpheres® in rat femoral vein and rabbit ear artery embolization models. Therefore the size-tunable and biodegradable thrombin-functionalized carboxymethyl chitin microspheres Thr@CMCHm-30 possess great potential for effective hemostasis and endovascular embolization.

Carboxymethyl chitin or chitosan for osteoinduction effect on the human periodontal ligament stem cells.

Human periodontal ligament stem cells (hPDLSCs) are seeding cells for tissue-engineered treatment of alveolar bone regeneration. To elucidate carboxymethyl chitosan (CMCTS) and carboxymethyl chitin (CMCT) effect on osteogenic differentiation, hPDLSCs were isolated and treated with CMCTS or CMCT. Cell viability and multiplication capacity were measured. The expression of classic osteogenic related molecules, including Alkaline Phosphatase (ALP), Phosphoprotein 1 (OPN), RUNX family transcription factor 2 (Runx2) and Osteocalcin (OCN), were determined. Mineralization levels were detected by Alizarin Red staining. Results showed that both CMCTS and CMCT treatment had the maximal promoting ability for hPDLSCs viability below the concentration of 100 µg/mL, while CMCTS improved hPDLSCs mineralization significantly. CMCTS induced multiple-factor high expression, including ALP, Runx2, OPN and OCN, whereas slightly osteoinductive bioactivity of CMCT was mainly due to ALP. Therefore, CMCTS had a more significant advantage for osteoinductive differentiation of hPDLSCs than CMCT, which may be a promising material for periodontal regeneration.

Towards the application of carboxymethyl chitin/ionic liquid blend as polymer electrolyte membrane for aqueous batteries.

Carboxymethyl chitin (CMChit) has the potential to be used as a solid polymer electrolyte (SPE) based on its ionic conductivity value of the order of 10-6 S·cm-1 in self-standing membranes. In controlled humidity of 65RH%, carboxymethyl chitin based membrane blended with 1-Butyl-3-methylimidazolium acetate (BMIM[Ac]) ionic liquid (IL) (40 wt%) showed a threshold value of ionic conductivity in the order of 10-4 S·cm-1 and electrochemical stability was up to 2.93 V. The effects of the relative humidity and ionic liquid weight fraction on the ionic conductivity and structural changes were investigated in detail. Furthermore, the X-ray diffraction (XRD) diffractogram indicated a clear reduction of crystallinity of the CMChit. The Field-emission scanning electron microscopy (FESEM) observation of the cross-sections confirmed the homogeneity of the prepared blend. This electrolyte was tested in symmetric cells based on Zn//SPE//Zn and showed good reversibility and potential for application in proton-conducting batteries.

Modified porous carboxymethyl chitin microspheres by an organic solvent-free process for rapid hemostasis.

Rapid and effective hemorrhage control is essential to reduce mortality following traumatic injuries. Herein we developed an organic solvent-free process to prepare carboxymethyl chitin microsphere (CMCHm) in an aqueous two-phase system through heating and freeze-drying. To further enhance the hemostatic performance of CMCHm, we loaded calcium ions and in-situ polymerized dopamine to get modified hemostatic microspheres CMCHm-Ca2+ and CMCHm-PDA, respectively. The size of these microspheres was mainly distributed between 50 µm and 150 µm, and the porous microstructure was observed by SEM. The data of in vitro degradation, cell cytotoxicity, and hemolysis test indicated good biocompatibility of these microspheres. Importantly, CMCHm-Ca2+ and CMCHm-PDA displayed better hemostatic performance compared with CMCHm and the positive controls Yunnan baiyao® and Quickclean®. Especially, the bleeding time was reduced to 59 s (CMCHm-Ca2+) and 45 s (CMCHm-PDA) in the femoral artery/vein cut model, respectively. All these demonstrate CMCHm-Ca2+ and CMCHm-PDA hold great potential for rapid hemostasis.

Multi-functional carboxymethyl chitin-based nanoparticles for modulation of tumor-associated macrophage polarity.

Current challenge of using cytokines is its poor distribution and systemic side effects. To avoid this issue, we prepared the tumor-targeted and microenvironment-responsive nanocarriers (TRN), which were consisted of α-tocopheryl succinate (α-TOS) loaded mesoporous silica nanoparticles as cores, and surface-modified by thioketal-linkage, electrostatically coated with carboxymethyl chitin, and further anchored glucose-regulated protein 78-binding peptide as shells for encapsulating IL-12. TRN showed a size of 260 nm after encapsulated IL-12 and α-TOS with loading content of 0.0206% and 7.21%, respectively, and exhibited good biocompatibility to 4 T1 cells and macrophages. Moreover, IL-12/α-TOS loaded TRN displayed obvious anti-tumor efficacy on BALB/c nude mice bearing 4 T1 tumors, which was derived from promoted targeting to tumor tissue, endocytosed by macrophages and locally release IL-12 to subsequently repolarize tumor-associated macrophages into tumoricidal M1 phenotype with reduced side effects. The nanosystem exhibited as a promising strategy with functional conversion of macrophages in tumor microenvironment for anti-tumor therapy.

Construction of hydrogels based on the homogeneous carboxymethylated chitin from Hericium erinaceus residue: Role of carboxymethylation degree.

Carboxymethyl chitin hydrogels with different degree of substitution (DS) were prepared by the homogeneous carboxymethylation of chitin extracted from Hericium erinaceus residue. The effect of DS on gel structure and property were studied. Results showed that the DS of carboxymethyl chitin hydrogels can be increased by increasing the amount of sodium chloroacetate. The equilibrium swelling degree and pH swelling sensitivity of the hydrogels were enhanced as the increase of DS. Zeta potential, low-field nuclear magnetic resonance, contact angle and molecular dynamics simulation results suggested that the introduction of carboxymethyl functional group enhanced the negative charge, water mobility, surface hydrophilicity and the ability to form hydrogen bonds with water of the hydrogels, resulting in an increased swelling degree of the hydrogels. Moreover, the prepared hydrogels showed different adsorption capability to various dyes, and the adsorption performance of the prepared hydrogels for cationic dyes could be enhanced as the increase of DS.

A new type of bilayer dural substitute candidate made up of modified chitin and bacterial cellulose.

In the field of neurosurgery, timely and effective repair of dura mater plays an important role in stabilizing the physiological functions of the human body. Therefore, the aim of this study is to develop a new type of bilayer membrane as a dural substitute candidate. It consists of a dense layer that prevents cerebrospinal fluid leakage and a porous layer that promotes tissue regeneration. The dense layer, a composite polysaccharid film, was composed of high molecular weight chitosan (CS) and bacterial cellulose (BC). The porous layer, a composite polysaccharid scaffold cross-linked by glutaraldehyde (GA) or citric acid (CA) respectively, was composed of O-carboxymethyl chitin (O-CMCH) and BC. The bilayer dural substitutes were characterized in terms of SEM, mechanical behavior, swelling rate, anti-leakage test, in vitro cytotoxicity, proliferation, and animal experiment. Results indicated that all prepared dural substitutes were tightly bound between layers without excessively large cavities. The porous layer showed appropriate pore size (90～200 µm) with high porous connectivity. The optimized bilayer dural substitutes showed suitable swelling rate and mechanical behavior. Furthermore, no leakage was observed during testing, no cytotoxicity effect on NIH/3T3 cells, and exhibited excellent cell proliferation promoting properties. Also, it was observed that it did not deform in the peritoneal environment of mice, and tissue inflammation was mild.

Preparation and analysis of photochromic behavior of carboxymethyl chitin derivatives containing spiropyran moieties.

1'-(2-Acryloxyethyl)-3,3'-dimethyl-6-nitrospiro[2 H-1-benzopyran-2,2'-indoline] (SPA) was synthesized and grafted onto a water-soluble carboxymethyl chitin (CMCH) macromolecule to prepare a photochromic copolymer (CMCH-g-SPA). The structure of CMCH-g-SPA was characterized by Fourier-transform infrared (FT-IR) spectroscopy, thermogravimetric (TG) analysis, X-ray diffraction (XRD) analysis, water-solubility evaluation, and UV-vis spectroscopy. XRD patterns of CMCH-g-SPA revealed that grafting copolymerization disrupts the CMCH semicrystalline structure, thus improving water solubility. UV-vis spectroscopy results supported the negative photochromic behavior of the merocyanine (MC) form of CMCH-g-SPA (CMCH-g-MCA) present in a water solution of the target copolymer. In addition to high solvent polarity, the intermolecular and intramolecular electrostatic attraction between the indolenine cation and the COO- anion were found to be influencing factors, which stabilize these MC form of spiropyran groups grafted onto CMCH. In a water solution, visible light bleaching was completed over a short period (8 minutes) under artificial visible light irradiation and the thermal coloration reaction, whose rate constant at 25 °C was 4.64 × 10-4 s-1, which fit the first-order reaction equation. After ten photochromic cycles in water solution, the relative absorption intensity of CMCH-g-MCA decreased by 7.92%.

Smart pH/magnetic sensitive Hericium erinaceus residue carboxymethyl chitin/Fe3O4 nanocomposite hydrogels with adjustable characteristics.

A smart hydrogel with pH/magnetic dual sensitivity was synthesized by in-situ synthesis of Fe3O4 inside carboxymethyl chitin hydrogel matrix prepared from Hericium erinaceus residue. The structure, pH/magnetic sensitivity, swelling and drug release behavior of the prepared hydrogels were investigated. The results showed that Fe3O4 nanoparticles were successfully synthesized and uniformly distributed within the hydrogels. The prepared hydrogels could be attracted by the magnet and exhibited sustained shrinkage behavior at low pH, with the desirable pH/magnetic sensitivity. The formed Fe3O4 could be developed inside the hydrogels by increasing the concentrations of precursor Fe2+/Fe3+ ions, and the magnetic sensitivity of hydrogels was enhanced, while the pH sensitivity and swelling degree were weakened. The Fe3O4 content-dependent behavior of the prepared hydrogels suggested the adjustable properties of hydrogels. The release of 5-Fu in simulated gastric and intestinal fluids followed the Fick diffusion mechanism and showed different release rates, indicating the pH-controlled drug release behavior.

A pH/ROS-responsive, tumor-targeted drug delivery system based on carboxymethyl chitin gated hollow mesoporous silica nanoparticles for anti-tumor chemotherapy.

A new nanosystem was prepared by coating ROS-cleavable thioketal (TK) bonded hollow mesoporous silica nanoparticles with carboxymethyl chitin via electrostatic interaction and further surface-anchored with glucose-regulated protein 78 binding peptide for targeted-delivery of doxorubicin (DOX) and α-tocopheryl succinate (α-TOS). The nanosystem (HMSN-TK-CMCH-GRP78P) showed an average size of 265 nm after loading DOX and α-TOS with a drug loading content of 4.06 % and 7.64 %, respectively. The in vitro release studies revealed the pH/ROS dual-responsibility of DOX/α-TOS loaded HMSN-TK-CMCH-GRP78P. The released α-TOS increased the intracellular ROS concentration, which could induce the cleavage of TK linkages and in turn accelerate DOX release. Moreover, the nanosystem could target to 4T1 cells, causing cell death in vitro and suppress tumor growth in vivo in 4T1-bearing BALB/c mice with reduced side effects, which illustrated that the nanosystem led to an effective anti-tumor efficacy and exhibited as a promising carrier to deliver chemotherapeutic agents for chemotherapy.

Degradable porous carboxymethyl chitin hemostatic microspheres.

Lethal hemorrhage could endanger lives. Although many hemostatic agents are commercially available with good clinical effect, it is necessary to develop novel hemostatic materials with high efficacy, biological safety, low cost, easily preparation and excellent biodegradability and biocompatibility. Here, novel biodegradable and uniform microspheres with regular spherical architecture and porous microstructure were prepared by thermosensitive carboxymethyl chitin (CMCH) with low substitution degree and low deacetylation through physical-crosslinking at high temperature without using any toxic crosslinkers. The obtained CMCH microspheres (CMCH-MS) were non-cytotoxic, low hemolytic potential and biodegradable in the presence of lysozyme. In vitro blood clotting evaluation indicated that the porous microsphere network structures and the hydrophilicity of the CMCH could promote hemostasis due to the quick blood absorption and local concentration of the coagulation factors for the CMCH-MS. The CMCH-MS showed much better hemorrhage control than negative control and traditional hemostatic chitosan, similar hemostatic performance as commercialized cross-linked starch microspheres in both rat tail and liver models. Thus, the new biodegradable porous CMCH-MS may hold great potential for hemorrhage control in medical treatment.

Regulation of the Morphological and Physical Properties of a Soft Tissue Scaffold by Manipulating DD and DS of O-Carboxymethyl Chitin.

To improve the biocompatibility/biodegradability as well as to lower the cost of the popular glycosaminoglycan/collagen scaffold, a monocomponent's polysaccharide scaffold based on biomimetic chemical modification of chitin from lower organisms was developed creatively. O-Carboxymethyl chitin (O-CMCH) was prepared by chloroacetic acid substitution of alkalized chitin. The cross-linked O-CMCH soft tissue scaffold was constructed by a sol-gel freeze-drying method. The key parameters of the O-CMCH molecular structure, the degree of deacetylation (DD), and the degree of substitution (DS) were used to regulate the morphology and physical properties of the scaffold. The optimized scaffolds were implanted subcutaneously in mice, and the inflammation reaction of surrounding tissues, dermal tissue growth, and scaffold degradation were observed dynamically by light microscopy and scanning electron microscopy. The results showed that the micropores of the scaffold constructed by O-CMCH with DD = 0.53 and DS = 0.61 were uniformly distributed and in communication with each other, and the pore size was 100-150 µm, with high porosity (93.52 ± 4.68%), high swelling ratio (1402 ± 70%), and high skeleton cross-linking degree (93.4 ± 4.6%). Its tensile strength reached 0.183 ± 0.009 MPa, and its elongation at break was 18.7 ± 0.9%. Furthermore, it could be degraded to less than 10% after 16 days in phosphate buffer solution (pH = 7.4) with 0.2 mg/mL lysozymes (≥ 20 000 U/mg). The early inflammation after implanting the optimized scaffolds in mice showed no difference compared with the control. The scaffold material induced dermal tissues to grow over it and was degraded gradually in vivo. The optimized scaffold regulated by DD and DS of O-CMCH possessed suitable morphology and physical properties for soft tissue engineering technology and exhibited a high applicable value.

Thermosensitive injectable in-situ forming carboxymethyl chitin hydrogel for three-dimensional cell culture.

Injectable hydrogels have gained great attentions for cell therapy and tissue regeneration as a result of the applications in minimally invasive surgical procedures with the ease of handling and complete filling of the defect area. Here, a novel biodegradable, thermosensitive and injectable carboxymethyl chitin (CMCH) hydrogel was developed for three-dimensional (3D) cell culture. The obtained CMCH solution remained transparent liquid flowing easily at low temperatures and gelled rapidly at 37°C. The gelation time of CMCH hydrogels could be easily tuned by varying temperature and the degree of carboxymethylation, which facilitates the cell encapsulation process at room temperature and in-situ forming hydrogel at body temperature. Moreover, the CMCH-14 hydrogels in PBS buffer remained stable and continuous porous structure and could be degraded in the presence of lysozyme or hyaluronidase. HeLa cells proliferated sustainably and self-assembled to form 3D multicellular spheroids with high cell activity on the surface of CMCH-14 hydrogel. Encapsulation of COS-7 cells within the in-situ forming CMCH hydrogel demonstrated that CMCH hydrogels promoted cell survival and proliferation. In vivo mouse study of the CMCH hydrogels showed good in-situ gel formation and tissue biocompatibility. Thus, the biodegradable thermosensitive injectable CMCH hydrogels hold potential for 3D cell culture and biomedical applications. STATEMENT OF SIGNIFICANCE: Biodegradable hydrogels have been widely studied for cell therapy and tissue regeneration. Herein, we report a novel thermosensitive injectable carboxymethyl chitin (CMCH) hydrogel for 3D cell culture, which was synthesized homogeneously from the bioactive natural chitin through the "green" process avoiding using organic solvent. The CMCH solutions exhibited rapid thermoresponsive sol-to-gel phase transition behavior at 37°C with controllable gelation times, which facilitates the cell encapsulation process at room temperature and in-situ forming hydrogel at body temperature. Importantly, in vitro 3D cell culture and in vivo mouse study of the CMCH hydrogel showed promotion of cell survival and proliferation, good in-situ gel formation and biocompatibility. We believe that such thermosensitive injectable CMCH hydrogels would be very useful for biomedical applications, such as tumor model for cancer research, post-operative adhesion prevention, the regeneration of cartilage and central nervous system and so on.

Synthesis of carboxymethyl chitin in aqueous solution and its thermo- and pH-sensitive behaviors.

Homogenous modification of natural chitin offers the advantage of fair structure control. In this work, novel carboxymethyl chitins (CMCHs) with broad range of degree of substitution (DS: 0.035 to 0.74), high degree of acetylation (DA) and little de-polymerization were synthesized homogeneously in aqueous NaOH/urea solution. The simultaneous determination of DA, DS and carboxymethylation fraction at C3 and C6 for these CMCHs was achieved by proton NMR in acidic deuterated aqueous solution for the first time. Due to the good homogeneity, the prepared CMCH-4 with lower DS of carboxymethylation exhibited, for the first time to our knowledge, dual thermo- and pH-sensitive properties. The nontoxic thermo-sensitive polymer systems gel at body temperature (37 °C) in physiological condition, which is very useful as injectable hydrogels for drug delivery and tissue engineering.

Preparation and characterization of cross-linked carboxymethyl chitin porous membrane scaffold for biomedical applications.

Porous dermal scaffold membrane (PDSM) was successfully prepared by using a so-called sol-gel freeze-drying method. In this method, the carboxymethyl chitin (CMC) hydrosol was first cross-linked by 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and then lyophilized to form the PDSM. For the first time, this research focused on the cross-linked CMC as the only component for three-dimensional PDSM. The effects of cross-linking conditions on the performance of the PDSM were investigated. And PDSM with optimal performance was obtained through 4-h cross-linking at 4 wt% of CMC concentration in the hydrosol, where the mass ratio of EDC to NHS to CMC was 5:3:10. The porosity of the optimized PDSM was more than 90% and the water swelling rate was above 4000%. The pore size was well distributed and was between 100 µm and 200 µm. And the tensile strength was above 0.09 MPa. The as-made PDSM could be degraded above 80% in 12 days in the presence of a 0.2mg/mL lysozyme solution. Very importantly, the PDSM had no cytotoxicity and good biocompatibility from MTT assays. Our results showed the application possibility of the as-prepared PDSM as dermal scaffold for skin tissue engineering.

Biological adhesive based on carboxymethyl chitin derivatives and chitin nanofibers.

Novel biological adhesives made from chitin derivatives were prepared and evaluated for their adhesive properties and biocompatibility. Chitin derivatives with acrylic groups, such as 2-hydroxy-3-methacryloyloxypropylated carboxymethyl chitin (HMA-CM-chitin), were synthesized and cured by the addition of an aqueous hydrogen peroxide solution as a radical initiator. The adhesive strength of HMA-CM-chitin increased when it was blended with chitin nanofibers (CNFs) or surface-deacetylated chitin nanofibers (S-DACNFs). HMA-CM-chitin/CNFs or HMA-CM-chitin/S-DACNFs have almost equal adhesive strength compared to that of a commercial cyanoacrylate adhesive. Moreover, quick adhesion and induction of inflammatory cells migration were observed in HMA-CM-chitin/CNF and HMA-CM-chitin/S-DACNF. These findings indicate that the composites prepared in this study are promising materials as new biological adhesives.

Secreted major Venus flytrap chitinase enables digestion of Arthropod prey.

Predation plays a major role in energy and nutrient flow in the biological food chain. Plant carnivory has attracted much interest since Darwin's time, but many fundamental properties of the carnivorous lifestyle are largely unexplored. In particular, the chain of events leading from prey perception to its digestive utilization remains to be elucidated. One of the first steps after the capture of animal prey, i.e. the enzymatic breakup of the insects' chitin-based shell, is reflected by considerable chitinase activity in the secreted digestive fluid in the carnivorous plant Venus flytrap. This study addresses the molecular nature, function, and regulation of the underlying enzyme, VF chitinase-I. Using mass spectrometry based de novo sequencing, VF chitinase-I was identified in the secreted fluid. As anticipated for one of the most prominent proteins in the flytrap's "green stomach" during prey digestion, transcription of VF chitinase-I is restricted to glands and enhanced by secretion-inducing stimuli. In their natural habitat, Venus flytrap is exposed to high temperatures. We expressed and purified recombinant VF chitinase-I and show that the enzyme exhibits the hallmark properties expected from an enzyme active in the hot and acidic digestive fluid of Dionaea muscipula. Structural modeling revealed a relative compact globular form of VF chitinase-I, which might contribute to its overall stability and resistance to proteolysis. These peculiar characteristics could well serve industrial purposes, especially because of the ability to hydrolyze both soluble and crystalline chitin substrates including the commercially important cleavage of α-chitin.