Coagulant Protein-Free Blood Coagulation Using Catechol-Conjugated Adhesive Chitosan/Gelatin Double Layer.

Since the discovery of polyphenolic underwater adhesion in marine mussels, researchers strive to emulate this natural phenomenon in the development of adhesive hemostatic materials. In this study, bio-inspired hemostatic materials that lead to pseudo-active blood coagulation, utilizing traditionally passive polymer matrices of chitosan and gelatin are developed. The two-layer configuration, consisting of a thin, blood-clotting catechol-conjugated chitosan (CHI-C) layer and a thick, barrier-functioning gelatin (Geln) ad-layer, maximizes hemostatic capability and usability. The unique combination of coagulant protein-free condition with CHI-C showcases not only coagulopathy-independent blood clotting properties (efficacy) but also exceptional clinical potential, meeting all necessary biocompatibility evaluation (safety) without inclusion of conventional coagulation triggering proteins such as thrombin or fibrinogen. As a result, the CHI-C/Geln is approved by the Ministry of Food and Drug Safety (MFDS, Republic of Korea) as a class II medical device. Hemostatic efficacy observed in multiple animal models further demonstrates the superiority of CHI-C/Geln sponges in achieving quick hemostasis compared to standard treatments. This study not only enriches the growing body of research on mussel-inspired materials but also emphasizes the potential of biomimicry in developing advanced medical materials, contributing a promising avenue toward development of readily accessible and affordable hemostatic materials.

Korean Amberjack Skin-Inspired Hyaluronic Acid Bioink for Reconstruction of Human Skin.

Decellularized extracellular matrix (dECM) has been extensively employed as tissue engineering scaffolds because its components can greatly enhance the migration and proliferation of cultivating cells. In this study, we decellularized Korean amberjack skin and incorporated soluble fractions in hyaluronic acid hydrogels with 3D-printed tissue engineering hydrogels to overcome any limitation of animal-derived dECM. The hydrolyzed fish-dECM was mixed with methacrylated hyaluronic acid and chemically crosslinked to 3D-printed fish-dECM hydrogels, where fish-dECM contents affected both printability and injectability of the hydrogels. Swelling ratios and mass erosion of the 3D-printed hydrogels were dependent on fish-dECM contents, where higher fish-dECM in the hydrogel increased swelling ratios and mass erosion rates. The higher content of fish-dECM considerably enhanced the viability of the incorporated cells in the matrix for 7 days. Artificial human skin was constructed by seeding human dermal fibroblasts and keratinocytes in the 3D-printed hydrogels, and a formation of a bilayered skin was visualized with tissue staining. Thus, we envision that 3D-printed hydrogels containing fish-dECM can be an alternative bioink composed of a non-mammal-derived matrix.

Clinical application of a new hemostatic material using mussel-inspired catecholamine hemostat: A pilot study.

BACKGROUNDS/AIMS: This study aimed to evaluate clinical application of InnoSEAL Plus (a mussel-inspired catecholamine hemostat) as a new hemostatic material for humans. METHODS: Patients treated with topical hemostatic patches after liver resection were enrolled. They were divided into an experimental group (InnoSEAL Plus group) and two control groups (TachoSil® group and Surgicel Fibrillar® group) for efficacy evaluation. RESULTS: A total of 15 patients were enrolled. Each group had five patients. The 3-minute hemostasis success rate was 80.0% (4/5 patients) in the InnoSEAL Plus group, 80.0% (4/5 patients) in the TachoSil® group, and 40.0% (2/5 patients) in the Surgicel Fibrillar® group, showing no significant difference in the success rate among these groups (p > 0.05). All three groups exhibited 100% success rate for 10-minute hemostasis. Both InnoSEAL Plus and TachoSil® groups had one patient developing adverse events, which were treated easily with drug administrations. CONCLUSIONS: InnoSEAL Plus is expected to be functionally not inferior to other conventional hemostatic agents. However, it is necessary to confirm this through multicenter prospective studies in the future.

A multicenter, prospective, randomized clinical trial of marine mussel-inspired adhesive hemostatic materials, InnoSEAL Plus.

PURPOSE: InnoSEAL Plus is an adhesive, coagulant-free hemostatic material that mimics the adhesion mechanism of marine mussels. This study reports on the safety and efficacy of InnoSEAL Plus for patients with hemorrhage after hepatectomy despite first-line hemostasis treatments. METHODS: This is a multicenter, prospective, single-blinded, randomized clinical trial involving 96 hepatectomy patients. TachoSil was used as a comparator group. Three-minute and 10-minute hemostatic success rates were monitored. Rebleeding rates were also observed. Safety was assessed by recording all novel undesirable symptoms. RESULTS: InnoSEAL Plus showed a 3-minute hemostasis rate of 100%, while TachoSil had a rate of 98.0% (48 of 49 patients), demonstrating that the 2 had similar hemostatic efficacies. The difference in efficacy between the test and comparator group was 2.04%, and the lower limit of the one-sided 97.5% confidence interval was -1.92%; as this is greater than the noninferiority limit of -23.9%, the 2 treatments were equivalent. Meanwhile, the 10-minute hemostatic success rate was the same in both groups (100%). No rebleeding occurred in either group. In the safety evaluation, 89 patients experienced adverse events (45 in the test group and 44 in the comparator group). The difference between the 2 groups was not significant. No death occurred after application of the test or comparator group product. CONCLUSION: Given that InnoSEAL Plus is a coagulation factor-free product, the hemostasis results are encouraging, especially considering that TachoSil contains a coagulation factor. InnoSEAL Plus was found to be a safe and effective hemostatic material for control of bleeding in hepatectomy patients.

Self-sealing hyaluronic acid-coated 30-gauge intravitreal injection needles for preventing vitreous and drug reflux through needle passage.

Self-sealing hyaluronic acid (HA)-coated self-sealing 30-gauge needles exhibiting instant leakage prevention of intravitreal humor and injected drug were developed in this study. Ninety New Zealand rabbits were used in this study. We assessed dye regurgitation in intravitreal ICG dye injections using HA-coated needles (HA needle group) and conventional needles (control group). Vitreous humor levels of anti-vascular endothelial growth factor (VEGF) were compared between groups one, three, and seven days after intravitreal bevacizumab (0.016 mL) injections. Expression levels of inflammatory cytokines in the aqueous humor and vitreous humor, including prostaglandin E2 (PGE2), interferon-γ, tumor necrosis factor-α, interleukin (IL)-1ß, IL-4, IL-6, IL-17, and IL-8, were compared between HA needle, control, and normal (in which intravitreal injection was not performed) groups following 12 intravitreal injections over a period of one week. In the HA needle group, HA remained at the injection site and blocked the hole after intravitreal injection. Dye regurgitation occurred significantly less frequently in the HA needle group (16.7%) than the control group (55.6%) after intravitreal ICG dye injection. Meanwhile, vitreous anti-VEGF levels were markedly higher in the HA needle group than the control group one and three days after intravitreal bevacizumab injections. After 12 intravitreal injections, expression levels of aqueous and vitreous IL-8 significantly increased in the control group compared to the HA needle and normal groups. Conversely, there were no significant differences in the expression of the other seven cytokines among the three groups. Intravitreal injections using HA-coated self-sealing 30-gauge needles can block the outflow of vitreous humor and drugs through the needle passage.

Endoscopic application of mussel-inspired phenolic chitosan as a hemostatic agent for gastrointestinal bleeding: A preclinical study in a heparinized pig model.

Marine mussels secrete adhesive proteins to attach to solid surfaces. These proteins contain phenolic and basic amino acids exhibiting wet adhesion properties. This study used a mussel-inspired hemostatic polymer, chitosan-catechol, to treat gastrointestinal bleeding caused by endoscopic mucosal resection in a heparinized porcine model. We aimed to evaluate the hemostatic efficacy and short-term safety of this wet adhesive chitosan-catechol. We used 15 heparinized pigs. Four iatrogenic bleeding ulcers classified as Forrest Ib were created in each pig using an endoscopic mucosal resection method. One ulcer in each pig was untreated as a negative control (no-treatment group). The other three ulcers were treated with gauze (gauze group), argon plasma coagulation (APC group), and chitosan-catechol hemostatic agent (CHI-C group) each. The pigs were sacrificed on Days 1, 5, and 10, and histological examination was performed (n = 5 per day). Rapid hemostasis observed at 2 min after bleeding was 93.3% (14/15) in the CHI-C group, 6.7% (1/15) in the no-treatment group, 13.3% (2/15) in the gauze group, and 86.7% (13/15) in the APC group. No re-bleeding was observed in the CHI-C group during the entire study period. However, a few re-bleeding cases were observed on Day 1 in the no-treatment, gauze, and APC groups and on Day 5 in the gauze and APC groups. On histological analysis, the CHI-C group showed the best tissue healing among the four test groups. Considering the results, chitosan-catechol is an effective hemostatic material with reduced re-bleeding and improved healing.

Coagulopathy-independent, bioinspired hemostatic materials: A full research story from preclinical models to a human clinical trial.

Since the first report of underwater adhesive proteins of marine mussels in 1981, numerous studies have reported mussel-inspired synthetic adhesive polymers. However, none of them have developed up to human-level translational studies. Here, we report a sticky polysaccharide that effectively promotes hemostasis from animal bleeding models to first-in-human hepatectomy. We found that the hemostatic material instantly generates a barrier layer that seals hemorrhaging sites. The barrier is created within a few seconds by in situ interactions with abundant plasma proteins. Therefore, as long as patient blood contains proper levels of plasma proteins, hemostasis should always occur even in coagulopathic conditions. To date, insufficient tools have been developed to arrest coagulopathic bleedings originated from genetic disorders, chronic diseases, or surgical settings such as organ transplantations. Mussel-inspired adhesion chemistry described here provides a useful alternative to the use of fibrin glues up to a human-level biomedical application.

Chitosan oral patches inspired by mussel adhesion.

Oral mucosal drug delivery systems have been developed to expedite the regeneration of oral mucosa, there are still many challenges related to residence time for drugs because the ceaseless changes of saliva, mouth movement, and involuntary swallowing prevent robust adhesion of drugs and/or drug-loaded biomaterials. Thus, it is highly desirable to develop the delivery platforms exhibiting robust, stable adhesion within oral cavities. Herein, we have developed an adhesive polysaccharide oral patch called 'Chitoral' that utilizes chemical principles shown in wet-resistant mussel adhesion. Chitoral plays an important role as an adhesive layer in wet environments. We unexpectedly found that Chitoral instantly dissolves upon contact with saliva and a labial mucous layer, and then the dissolved Chitoral compounds forms an insoluble adhesion layer with mucins at Chitoral/mucous interface nearly immediate actions. Later, Chitoral gradually converts into adhesive hydrogels by the cooperative actions of covalent crosslinking and physical entanglement. The instant, robust muco-adhesion properties of Chitoral provides long-lasting therapeutic effects of drugs resulting enhanced healing of oral ulcer. Thus, mussel-inspired, mucous-resistant adhesive platforms, Chitoral, can be a platform for oral mucosal drug delivery systems.

Hemostatic Ability of Chitosan-Phosphate Inspired by Coagulation Mechanisms of Platelet Polyphosphates.

Hemostatic materials have been studied to minimize bleeding time. Recently, polyphosphate (polyP) have received attention as potential hemostatic compounds, which are released from activated platelets. Long polyP chains are essential to form thick fibrin clots. Herein, chitosan is functionalized by covalently tethering phosphate groups to mimic polyP. It is hypothesized that utilizing a known hemostatic polysaccharide, chitosan, and tethering phosphate groups to mimic polyP's functionality show synergistic effect in hemostasis. Five different phosphorylated chitosan conjugates (Chi-Ps), s-Chi-7P, s-Chi-28P, s-Chi-74P, is-Chi-29P, and is-Chi-56P are prepared, where "s" indicates water soluble Chi-Ps and "is" represents water insoluble Chi-Ps. Unexpectedly, an important carbon in D-glucosamine is found, which determines chitosan solubility. Phosphate groups conjugated to C6 carbon resulted in water soluble Chi-P, but conjugation to C3 group exhibited water insoluble behavior. Hemostasis capability showed a positive correlation with the degree of phosphate conjugations regardless of water solubility of Chi-P.

Hemostatic Swabs Containing Polydopamine-like Catecholamine Chitosan-Catechol for Normal and Coagulopathic Animal Models.

All animal experiments for evaluating drug efficacy or developing medical devices are unavoidably accompanied by bleeding that result in unreliable outcomes with large variations between individuals. Herein, we developed hemostatic swabs prepared by a mussel-inspired catecholamine polymer called chitosan-catechol, which was inspired by the chemical composition of the well-known material-independent coating material of polydopamine. The hemostatic ability of the swabs resulted from the formation of self-sealing membranes by rapid intermolecular interactions between whole blood proteins and the applied chitosan-catechol. The blood protein/chitosan-catechol composite sealing membrane resulted in dramatic decreases in bleeding for both normal and coagulopathic models, such as diabetes.

Targeting protein and peptide therapeutics to the heart via tannic acid modification.

Systemic injection into blood vessels is the most common method of drug administration. However, targeting drugs to the heart is challenging, owing to its dynamic mechanical motions and large cardiac output. Here, we show that the modification of protein and peptide therapeutics with tannic acid-a flavonoid found in plants that adheres to extracellular matrices, elastins and collagens-improves their ability to specifically target heart tissue. Tannic-acid-modified (TANNylated) proteins do not adsorb on endothelial glycocalyx layers in blood vessels, yet they penetrate the endothelium to thermodynamically bind to myocardium extracellular matrix before being internalized by myoblasts. In a rat model of myocardial ischaemia-reperfusion injury, TANNylated basic fibroblast growth factor significantly reduced infarct size and increased cardiac function. TANNylation of systemically injected therapeutic proteins, peptides or viruses may enhance the treatment of heart diseases.

Complete prevention of blood loss with self-sealing haemostatic needles.

Bleeding is largely unavoidable following syringe needle puncture of biological tissues and, while inconvenient, this typically causes little or no harm in healthy individuals. However, there are certain circumstances where syringe injections can have more significant side effects, such as uncontrolled bleeding in those with haemophilia, coagulopathy, or the transmission of infectious diseases through contaminated blood. Herein, we present a haemostatic hypodermic needle able to prevent bleeding following tissue puncture. The surface of the needle is coated with partially crosslinked catechol-functionalized chitosan that undergoes a solid-to-gel phase transition in situ to seal punctured tissues. Testing the capabilities of these haemostatic needles, we report complete prevention of blood loss following intravenous and intramuscular injections in animal models, and 100% survival in haemophiliac mice following syringe puncture of the jugular vein. Such self-sealing haemostatic needles and adhesive coatings may therefore help to prevent complications associated with bleeding in more clinical settings.

PEGylation and HAylation via catechol: α-Amine-specific reaction at N-terminus of peptides and proteins.

UNLABELLED: The development of chemoselective, site-specific chemistries for proteins/peptides is essential for biochemistry, pharmaceutical chemistry, and other fields. In this work, we found that catechol, which has been extensively utilized as an adhesive molecule for material-independent surface chemistry and as a crosslinker in hydrogel preparation, specifically reacts with N-terminal α-amines, avoiding the ε-amine group in lysine. A conjugate of methoxy-poly(ethylene glycol)-catechol called mPEG-cat chemoselectively reacts with N-terminal amine groups at neutral pH resulting in site-specific PEGylation. To demonstrate the versatility of this catechol chemoselective reaction, we used four proteins (lysozyme, basic-fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), insulin, and erythropoietin (EPO)) as well as two peptides (hinge-3 and laminin-derived peptide (LDP)). All the tested macromolecules showed N-terminal site-specific modifications. Furthermore, we prepared another catechol grafted conjugate called hyaluronic acid-catechol (HA-cat) to demonstrate that this catechol-involved chemoselective chemistry is not specific for PEG conjugates. This new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for a variety of purposes. STATEMENT OF SIGNIFICANCE: Considering the fact that biological activities of proteins or peptides depend largely on their 3-dimensional conformation, the orientation-controllable reaction is very important for preserving the intrinsic functionality of them. In addition to PEG, many other bio-polymers such as oligonucleotides, antibodies, and oligosaccharides have been conjugated with proteins or peptides for various biomedical applications. Although several chemoselective conjugation chemistries have been reported, conjugation efficiencies are different depending on types of proteins or polymers, and thus there've been strong needs for the development of alternative strategy of chemoselective conjugation that can be applied for a variety of therapeutic proteins towards high biological activities. We are certain this new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for various purposes.

Fine-scale map of encyclopedia of DNA elements regions in the Korean population.

The International HapMap Project aims to generate detailed human genome variation maps by densely genotyping single-nucleotide polymorphisms (SNPs) in CEPH, Chinese, Japanese, and Yoruba samples. This will undoubtedly become an important facility for genetic studies of diseases and complex traits in the four populations. To address how the genetic information contained in such variation maps is transferable to other populations, the Korean government, industries, and academics have launched the Korean HapMap project to genotype high-density Encyclopedia of DNA Elements (ENCODE) regions in 90 Korean individuals. Here we show that the LD pattern, block structure, haplotype diversity, and recombination rate are highly concordant between Korean and the two HapMap Asian samples, particularly Japanese. The availability of information from both Chinese and Japanese samples helps to predict more accurately the possible performance of HapMap markers in Korean disease-gene studies. Tagging SNPs selected from the two HapMap Asian maps, especially the Japanese map, were shown to be very effective for Korean samples. These results demonstrate that the HapMap variation maps are robust in related populations and will serve as an important resource for the studies of the Korean population in particular.

Initial transcriptome and proteome analyses of low culture temperature-induced expression in CHO cells producing erythropoietin.

Low culture temperature is known to enhance the specific productivity of Chinese hamster ovary (CHO) cells expressing erythropoietin (EPO) (LGE10-9-27). Genomic and proteomic approaches were taken to better understand the intracellular responses of these CHO cells resulting from use of low culture temperature (33 degrees C). For transcriptome analysis, commercially available rat and mouse cDNA microarrays were used. The data obtained from the rat and mouse cDNA chips were only somewhat informative in understanding the gene expression profile of CHO cells because of their different sequence homologies with CHO transcriptomes. Overall, transcriptome analysis revealed that low culture temperature could lead to changes in gene expression in various cellular processes such as metabolism, transport, and signaling pathways. Proteome analysis was carried out using 2-D PAGE. Based on spot intensity, 60 high intensity protein spots, from a total of more than 800, were chosen for MS analysis. Forty of the 60 protein spots, which represent 26 different kinds of proteins, were identified by MALDI-TOF-MS and validated by MS/MS. Compared to the reference temperature (37 degrees C), the expression levels of seven proteins (PDI, vimentin, NDK B, ERp57, RIKEN cDNA, phosphoglycerate kinase, and heat shock cognate 71 kDa protein) were increased over twofold at 33 degrees C and those of two proteins (HSP90-beta and EF2) were decreased over twofold at 33 degrees C. Taken together, the results demonstrate the potential of combined analysis of transcriptome and proteome analyses as a tool for the systematic comprehension of cellular mechanisms in CHO cells.

Proteome analysis of antibody-expressing CHO cells in response to hyperosmotic pressure.

To better understand intracellular responses to hyperosmotic pressure of recombinant Chinese hamster ovary (rCHO) cells expressing an antibody, we have taken a proteomics approach. Using two-dimensional electrophoresis and mass spectrometry, a proteome profile of rCHO cells comprising 23 identified proteins was established. On the basis of this proteome profile, we found three proteins of which expression levels were significantly changed at 450 mOsm/kg. Compared to the results at 300 mOsm/kg, two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, were found to be up-regulated, probably leading to an increased metabolic energy for antibody synthesis. The elevation of specific glucose consumption rate at 450 mOsm/kg agreed with the up-regulation of these glycolytic enzymes. On the other hand, tubulin expression was down-regulated, reflecting a depressed cell growth rate at 450 mOsm/kg. Taken together, this study shows the potential of the proteomics approach in understanding intracellular and physiological changes in cells and seeking a better insight into possible environmental or genetic manipulation approaches for increasing foreign protein production in rCHO cells.