天然多糖醛改性抗菌快速吸收止血海绵-动脉网

Antibacterial and rapidly absorbable hemostatic sponge by aldehyde modification of natural polysaccharide

Nature 等信源发布 2024-07-21 23:26

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AbstractMassive hemorrhage following tissue trauma has high mortality owing to the lack of timely intervention. However, research on utilizing hemostats for humans is limited; therefore, developing an efficient emergency hemostatic agent is imperative. We developed a hemostatic sponge using natural polysaccharide riclin, theoretically modified with 50% aldehyde content (AR50).

摘要由于缺乏及时干预，组织创伤后大出血死亡率很高。然而，对人类使用止血剂的研究是有限的；因此，开发一种有效的紧急止血剂势在必行。我们使用天然多糖riclin开发了一种止血海绵，理论上用50%醛含量（AR50）修饰。

The AR50 sponge, with quasi-honeycomb channels and appropriate aldehyde content, exhibits ultra-high blood absorption (59.4 g·g−1) and rapidly targets erythrocytes and platelets to form a stable barrier. It surpasses most commercial hemostats in porcine artery scission (reducing hemostasis time and blood loss by 53 s and 4.2 g), hepatic bleeding laceration (68 s and 2.6 g), and perforation models (140 s and 4.9 g).

AR50海绵具有准蜂窝状通道和适当的醛含量，具有超高的血液吸收（59.4 g·g-1），并迅速靶向红细胞和血小板以形成稳定的屏障。它超过了猪动脉断裂（止血时间和失血量分别减少53 s和4.2 g），肝出血撕裂（68 s和2.6 g）和穿孔模型（140 s和4.9 g）中大多数商业止血剂。

The AR50 sponge is easily removed post hemostasis, exhibits antibacterial properties by destroying bacterial cell walls, and is safely absorbed by day 5, making it an ideal emergency hemostatic agent for massive hemorrhages in humans..

AR50海绵在止血后很容易去除，通过破坏细菌细胞壁表现出抗菌性能，并在第5天安全吸收，使其成为治疗人类大出血的理想紧急止血剂。。

IntroductionMilitary conflicts, accidents, and clinical surgeries resulting in severe tissue trauma can lead to uncontrollable hemorrhaging and wound infection, pivotal factors that may contribute to mass fatalities1,2. Ideal hemostatic agents for emergencies, battlefield scenarios, and pre-hospital situations should possess characteristics such as rapid hemostasis, anti-infection properties, easy removal, promotion of healing, stability, affordability, and biocompatibility3,4.

引言导致严重组织创伤的军事冲突，事故和临床手术可能导致无法控制的出血和伤口感染，这是可能导致大规模死亡的关键因素1,2。用于紧急情况，战场场景和院前情况的理想止血剂应具有快速止血，抗感染特性，易于去除，促进愈合，稳定性，可负担性和生物相容性等特征3,4。

In clinical applications, achieving hemostasis poses a significant challenge owing to high blood flow3,5 and is currently addressed with the use of a high-expansion sponge4,5. Sponges, with porous structures and liquid absorption capacities, based on characteristics such as shape memory, light weight, portability, stability, and ease of operation, sponges offer advantages in treating moderate and severe arterial and visceral bleeding3,5.Polysaccharides have gained attention as clinical frontier hemostatic materials owing to their outstanding biological activities, abundant sources, high biosafety levels, and easily modifiable structures3,6,7,8.

在临床应用中，由于高血流量3,5，实现止血是一个重大挑战，目前正在使用高膨胀海绵4,5来解决。海绵具有多孔结构和液体吸收能力，基于形状记忆，重量轻，便携性，稳定性和易操作性等特点，海绵在治疗中重度动脉和内脏出血方面具有优势3,5。多糖作为临床前沿止血材料因其优异的生物活性，丰富的来源，高生物安全水平和易于修饰的结构而备受关注3,6,7,8。

Polysaccharides and their derivatives, including dextran (Bloxx®), cellulose (Surgicel®, BloodSTOP®), alginate (Algosteril®, KALTOSTAT®), chitosan (HemCon®, Celox®, Chito SAMTM 100), and starch (TraumaDEX®), are widely used in preparing commercial hemostats3,4,8. While most polysaccharide-based hemostatic agents exhibit excellent biological safety, they face challenges such as low hemostatic activity, a short shelf life, a high contamination risk, low coagulation and anti-infection capacities, limiting their application in treating massive bleeding in clinical settings4,9.

多糖及其衍生物，包括葡聚糖（Bloxx®），纤维素（Surgicel®，BloodSTOP®），藻酸盐（Algosteril®，KALTOSTAT®），壳聚糖（HemCon®，Celox®，Chito SAMTM 100）和淀粉（TraumaDEX®），广泛用于制备商业止血剂3,4,8。虽然大多数基于多糖的止血剂表现出优异的生物安全性，但它们面临着诸如止血活性低，保质期短，污染风险高，凝血和抗感染能力低等挑战，限制了它们在临床环境中治疗大出血的应用4,9。

Regrettably, limited research has been reported on effective polysaccharide hemostat.

遗憾的是，关于有效多糖止血剂的研究报道有限。

(1)

The speed of liquid absorption was determined by calculating the slope of the initial 5 s of the NS or blood absorptive capacity versus time curve10,32.Blood samples for in vitro experimentsAll of the fresh blood used in coagulation experiments was obtained from healthy human volunteers, in accordance with the Regulations of the People’s Republic of China on the Administration of Blood Products, Nuremberg Code, and Ethical Principles of Human Experimentation.

液体吸收的速度是通过计算NS或血液吸收能力的初始5 s与时间曲线10,32的斜率来确定的。用于体外实验的血液样本凝血实验中使用的所有新鲜血液均来自健康人体志愿者，符合中华人民共和国血液制品管理条例，纽伦堡法规和人体实验伦理原则。

Informed written consents were obtained from volunteers. Approval was granted by the Ethics Committee of Bengbu Medical College (BBMCEC2020025).In vitro calcification clotting timeTen milligrams of AR sponge were added to a test tube, followed by immediate mixing of 1 mL of blood (ACD-anticoagulant) and 80 µL of CaCl2 (0.25 M).

从志愿者那里获得了知情的书面同意。蚌埠医学院伦理委员会（BBMCEC2020025）批准。体外钙化凝血时间将10毫克AR海绵加入试管中，然后立即混合1毫升血液（ACD抗凝剂）和80微升CaCl2（0.25M）。

The mixture was then shaken gently for 5 s. The test tube was tilted to the horizontal plane at an angle of 60° every 2–3 s until the liquid level stopped changing and the blood clotting time was determined.Whole-blood clotting kineticsA 25 mg sponge sample was positioned in the center of the petri dish and treated with a mixture of 250 µL of blood (ACD-anticoagulant) and 25 µL of CaCl2 (0.2 M) at 37 °C.

然后将混合物轻轻摇动5秒。将试管每2-3秒以60°的角度倾斜至水平面，直到液位停止变化并确定凝血时间。全血凝固动力学将25mg海绵样品置于培养皿的中心，并在37℃下用250μL血液（ACD抗凝剂）和25μlCaCl2（0.2M）的混合物处理。

This was followed by the addition of 20 mL of deionized water to ensure complete submersion at various periods (0, 15, 30, 60, 120, 180, and 240 s). Unbound blood cells were lysed by setting the shaker at 60 rpm for 10 min. The absorbance of the lysed cell solution (optical density [OD] experiment) was determined at 540 nm using a microplate reader (BioTek Epoch), with deionized water and the whole-blood mixture serving as the control (OD control).

然后加入20毫升去离子水，以确保在不同时期（0,15,30,60120180和240秒）完全浸没。通过将振荡器置于60 rpm下10分钟来裂解未结合的血细胞。使用酶标仪（BioTek Epoch）在540 nm处测定裂解细胞溶液的吸光度（光密度[OD]实验），去离子水和全血混合物作为对照（OD对照）。

The BCI was then calculated according to the following equation36:$${{\rm{BCI}}}\,( \% )=\frac{{{{\rm{OD}}}}_{{{\rm{experiment}}}.

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(2)

To further observe the process of the blood cells changing on the sponge, 20 µL of blood was applied to 2 mg of the AR50 sponge and washed thrice with PBS at specified time points. The sample was then fixed in 2.5% glutaraldehyde for 15 min, followed by a sequential dehydration process with alcohol solutions of increasing strengths (30%, 50%, 70%, 90%, 100%) for 5 min each time.

为了进一步观察血细胞在海绵上的变化过程，将20μL血液施加到2mg AR50海绵上，并在指定的时间点用PBS洗涤三次。然后将样品在2.5%戊二醛中固定15分钟，然后用强度增加的酒精溶液（30%，50%，70%，90%，100%）进行连续脱水过程，每次5分钟。

After natural drying, the blood contact interface was observed using SEM, with spongy riclin serving as the control.Thromboelastography (TEG) analysisTEG was determined using the Thrombelastography Analyzer (CFMSLEPU-8800)24,37. Owing to the specificity of the TEG test, no kaolin activator was added, and the powdered sample was pre-made.

自然干燥后，使用SEM观察血液接触界面，以海绵状riclin作为对照。血栓弹力图（TEG）分析使用血栓弹力图分析仪（CFMSLEPU-8800）24,37测定TEG。由于TEG测试的特异性，不添加高岭土活化剂，并预先制备粉末样品。

Subsequently, 340 µL of blood (ACD-anticoagulant) was homogeneously mixed with 5 mg of the sample in the TEG test cup at 37 °C, followed by the addition of 20 µL of 0.25 M CaCl2. Finally, the sample cups were loaded for measurement.PPP analysisPlasma clotting kinetics were determined, as previously reported22,34.

随后，将340μL血液（ACD抗凝剂）与5 mg样品在37℃下在TEG测试杯中均匀混合，然后加入20μl0.25M CaCl2。最后，装入样品杯进行测量。PPP分析如先前报道22,34，测定血浆凝血动力学。

Specifically, in a 96-well plate, 60 µL AR (2 wt.%) was evenly mixed with 60 µL of PPP, followed by adding and mixing 60 µL of 0.25 M CaCl2. The absorbance of the plasma mixture was recorded at 405 nm every minute for 1 h, and a dynamic curve was drawn. The plasma procoagulant efficiency of each group was compared according to the trends of their kinetic curves and the slopes of their linear range.Erythrocyte adhesion and morphologyThe blood (ACD-anticoagulant) was centrifuged at 200 × g for 10 min at room temperature, the plasma was replaced with an equal volume of PBS, and subsequently, the erythrocytes were obtained; approximately 45 µL of these cells were added to 5 mg of the sponge and incubated in a 24-well plate at 37 °C for 15 mi.

具体而言，在96孔板中，将60μlAr（2重量%）与60μlPPP均匀混合，然后加入60μl0.25M CaCl2并混合。在每分钟405nm处记录血浆混合物的吸光度1小时，并绘制动态曲线。。红细胞粘附和形态将血液（ACD抗凝剂）在室温下以200×g离心10分钟，用等体积的PBS代替血浆，随后获得红细胞；将大约45μL这些细胞加入到5mg海绵中，并在24孔板中于37℃温育15分钟。

(3)

Rabbit skin irritation testMultiple topical applications of the AR solution (2 wt.%) were administered to assess skin irritation. The dorsal skin was shaved, exposed (5 × 5 cm), and sterilized using UV light before application. A 1 mL solution of the materials was applied to the exposed skin three times a day at 2 h intervals.

。将背部皮肤剃毛，暴露（5×5厘米），并在施用前使用紫外线灭菌。将1mL材料溶液以2小时的间隔每天三次施用于暴露的皮肤。

The condition of the sensitized skin was continuously observed and histopathologically examined after 3 days.Cytotoxicity and cellular ROS assayNIH3T3 cells were incubated with PBS and AR50 (100, 200, 400, 800 µg) for 24 h, respectively. The cells were fixed and stained using Actin-Tracker Green and 4ʹ,6-diamidino-2-phenylindole (DAPI).

。细胞毒性和细胞ROS测定将NIH3T3细胞分别与PBS和AR50（100200400800μg）孵育24小时。将细胞固定并使用肌动蛋白追踪器Green和4'，6-二脒基-2-苯基吲哚（DAPI）染色。

The antioxidant effect of riclin with different degrees of oxidation was evaluated using ROS probes and ROS detection kits. NIH3T3 cells were treated as described above. The extracellular ROS scavenging of different groups was initially detected using DCFH-DA. The intracellular ROS scavenging ability of AR was then measured with an ROS assay kit.

使用ROS探针和ROS检测试剂盒评估具有不同氧化程度的riclin的抗氧化作用。如上所述处理NIH3T3细胞。首先使用DCFH-DA检测不同组的细胞外ROS清除率。然后用ROS测定试剂盒测量AR的细胞内ROS清除能力。

The images of samples were obtained using a Zeiss LSM880 confocal laser-scanning microscope, with constant settings maintained within the same experimental batch.Statistical analysisData from at least three samples per experiment are expressed as means ± standard deviation; statistical significance was set at P < 0.05.

使用Zeiss LSM880共聚焦激光扫描显微镜获得样品的图像，并在同一实验批次内保持恒定设置。统计分析每个实验至少三个样本的数据表示为平均值±标准偏差；统计学显着性设定为P<0.05。

Statistical analyses were performed using one-way or two-way analysis of variance and Tukey’s post-hoc test, using GraphPad Prism 6.0 and Origin Lab 8.0 software.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article..

使用GraphPad Prism 6.0和Origin Lab 8.0软件，使用单向或双向方差分析和Tukey事后检验进行统计分析。报告摘要有关研究设计的更多信息，请参阅本文链接的Nature Portfolio Reporting Summary。。

Data availability

The data supporting the results of this study are available from the corresponding author on reasonable request.

支持本研究结果的数据可根据合理要求从通讯作者处获得。

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Download referencesAcknowledgementsWe extend our gratitude to Dr. Jianfa Zhang for providing the riclin exopolysaccharides. This work received primary support from a research project of Anhui Provincial Key Research and Development Project (No. 202104j07020017), Natural Science Research Project of Anhui Educational Committee (No.

下载参考文献致谢我们感谢张建发博士提供riclin胞外多糖。这项工作得到了安徽省重点研究发展项目（编号202104J0702017）研究项目，安徽省教委自然科学研究项目（编号202104J0702017）的初步支持。

2022AH030139, No. 2022AH051498, No. 2023AH051923), Natural Science Foundation of Anhui Province (No. 2308085QH251), and National Natural Science Foundation of China (No. 32000822).Author informationAuthor notesThese authors contributed equally: Jinrun Zhang, Zenghui Chen, Dejie Zeng.Authors and AffiliationsDepartment of Physiology, Bengbu Medical University, Bengbu, 233030, ChinaJinrun Zhang, Zenghui Chen, Yuman Xia, Yizhuo Fan, Xinyu Zhang, Nan Li, Xiaofen Liu, Xiaqing Sun, Junhao Liu & Qi SunAnhui Province Key Laboratory of Immunology in Chronic Diseases, Bengbu Medical University, Bengbu, 233030, ChinaJinrun Zhang & Qi SunDepartment of Critical Care Medicine, The First Affiliated Hospital of Bengbu Medical University, Bengbu, 233004, ChinaDejie Zeng & Shibing ZhaoCenter for Molecular Metabolism, Nanjing University of Science & Technology, Nanjing, 210094, ChinaJianfa ZhangAuthorsJinrun ZhangView author publicationsYou can also search for this author in.

2022AH030139，编号2022AH051498，编号2023AH051923），安徽省自然科学基金（编号2308085QH251）和国家自然科学基金（编号3200822）。作者信息作者注意到这些作者做出了同样的贡献：张金润，陈增辉，曾德杰。作者及所属单位蚌埠医科大学生理学系，蚌埠，233030，中国张金润，陈增辉，夏玉满，范一卓，张新宇，李楠，刘晓芬，孙夏青，刘俊浩，齐孙楠安徽省慢性病免疫学重点实验室，蚌埠医科大学，233030，中国张金润，齐孙楠蚌埠医科大学第一附属医院重症医学科，233004，南京科技大学分子代谢中心，南京，210094，中国张建发作者张金润查看作者出版物您也可以在中搜索此作者。

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PubMed Google ScholarContributionsJ.Z. (Jinrun Zhang), Q.S., and J.L. conceived the project, designed the experiments, and wrote the paper; J.Z. (Jinrun Zhang), Z.C., and D.Z. conducted the synthesis and characterization of the samples; Z.C., X.Z., and N.L. obtained scanning electron microscopy and transmission electron microscopy images; J.Z.

PubMed谷歌学术贡献。Z、（张金润），Q.S。和J.L.构思了该项目，设计了实验并撰写了论文；J、 Z.（张金润），Z.C。和D.Z.进行了样品的合成和表征；Z、 C.，X.Z。和N.L.获得了扫描电子显微镜和透射电子显微镜图像；J、 Z。

(Jinrun Zhang), J.L., Y.X., and Y.F. carried out all the in vitro and in vivo experiments; D.Z., Y.X., and J.Z. (Jinrun Zhang) performed the antibacterial experiments; Q.S. and J.Z. (Jianfa Zhang) revised the draft; Y.F., X.L., X.S., S.Z., and J.Z. (Jianfa Zhang) analyzed the data and contributed to interpretation of the results.

（张金润），J.L.，Y.X。和Y.F.进行了所有的体外和体内实验；D、 Z.，Y.X。和J.Z。（张金润）进行了抗菌实验；Q、 S.和J.Z.（张建发）修订了草案；Y、 F.，X.L.，X.S.，S.Z。和J.Z.（张建发）分析了数据，并为结果的解释做出了贡献。

All authors discuss and approved the final manuscript.Corresponding authorsCorrespondence to.

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Communications Materials thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Steven Caliari and Jet-Sing Lee. A peer review file is available.

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Reprints and permissionsAbout this articleCite this articleZhang, J., Chen, Z., Zeng, D. et al. Antibacterial and rapidly absorbable hemostatic sponge by aldehyde modification of natural polysaccharide.

转载和许可本文引用本文Zhang，J.，Chen，Z.，Zeng，D。等人。通过天然多糖的醛改性抗菌和快速可吸收止血海绵。

Commun Mater 5, 129 (2024). https://doi.org/10.1038/s43246-024-00579-0Download citationReceived: 10 March 2024Accepted: 17 July 2024Published: 21 July 2024DOI: https://doi.org/10.1038/s43246-024-00579-0Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

Commun Mater 5129（2024）。https://doi.org/10.1038/s43246-024-00579-0Download引文收到日期：2024年3月10日接受日期：2024年7月17日发布日期：2024年7月21日OI：https://doi.org/10.1038/s43246-024-00579-0Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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