Platelet-camouflaged silver nanoparticle hydrogel accelerates wound healing in type 1 diabetic rats

doi:10.12307/2024.450

Abstract

Abstract: BACKGROUND: Bacterial infections and impaired angiogenesis have been obstacles to diabetic wound healing, and the problem of multidrug resistance cannot be ignored, so there is an urgent need to find a new therapeutic strategy.
OBJECTIVE: To prepare platelet-camouflaged silver nanoparticle hydrogel and observe its therapeutic effect on diabetic wounds.
METHODS: (1) In vitro test: The ultra-small silver nanoparticles (usAgNPs) were obtained by reflux heating. usAgNPs-PL/CMC hydrogel was prepared by coupling it with platelets (PL) and adding it to the carboxymethyl cellulose (CMC) hydrogel. The microstructures, swelling properties, and mechanical properties of the hydrogel were characterized. The antibacterial activity of the hydrogel was tested by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide method, reactive oxygen species method, bacterial biofilm method, and bacterial activity. (2) In vivo test: Forty SD rats were randomly divided into sham operation group, model group, CMC hydrogel group, usAgNPs/CMC hydrogel group, and usAgNPs-PL/CMC hydrogel group, with eight rats in each group. The type 1 diabetes model was established in the other four groups except the sham operation group. After successful model establishment, a skin wound (20 mm in diameter) deep to the fascia layer was made on the back of rats of the five groups. Rats in the sham operation group and model group were injected with normal saline. The rats in the CMC hydrogel group, usAgNPs/CMC hydrogel group, and usAgNPs-PL/CMC hydrogel group were implanted with the corresponding hydrogel, separately. The wound healing condition and healing quality were observed after 14 days of treatment.
RESULTS AND CONCLUSION: (1) In vitro test: The usAgNPs-PL/CMC hydrogel had a uniform three-dimensional network structure, good mechanical properties, and strong water absorption ability, which could remarkably inhibit the growth of Escherichia coli and Staphylococcus aureus, and had a good inhibition and clearance effect on the formation of biofilms of the two bacteria, and had a good antibacterial ability. (2) In vivo test: After 14 days of treatment, the wounds of the usAgNPs-PL/CMC hydrogel group were basically closed. The wound healing rate was the highest, and the number of wound colonies was significantly lower than that of the model group. After 14 days of treatment, hematoxylin-eosin and Masson staining showed that the tissue structure of regenerated dermis appeared on the wounds of the usAgNPs-PL/CMC hydrogel group, usAgNPs/CMC hydrogel group, and CMC hydrogel group, and the epidermis of the new granulation tissue was complete and thick. Parallel mature collagen fibers were observed in the usAgNPs-PL/CMC hydrogel group. After 7 days of treatment, CD31 immunohistochemistry and Ki67 immunofluorescence staining showed that the usAgNPs-PL/CMC hydrogel group had the highest number of microvessels and fibroblasts. (3) The results showed that usAgNPs-PL/CMC hydrogel could accelerate wound healing in type 1 diabetic rats by rapidly inhibiting bacteria and promoting angiogenesis.

Key words: ultra-small silver nanoparticle, platelet, carboxymethyl cellulose, hydrogel, diabetic wound, wound healing, bacterial inhibition

CLC Number:

Wang Jinlei, Li Ke, Zhao Liang. Platelet-camouflaged silver nanoparticle hydrogel accelerates wound healing in type 1 diabetic rats[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(17): 2659-2666.

Figures/Tables 13

生物膜的产生是导致抗生素耐药性和治疗效果差的原因之一，作者认为usAgNPs-PL/CMC水凝胶对生物膜具有高渗透性，对大肠杆菌和金黄色葡萄球菌的生物膜形成具有良好的抑制和清除作用，因此实验采用结晶紫染色和XTT测定两种方法来评价细菌生物膜的形成。XTT定量分析结果显示，usAgNPs-PL/CMC水凝胶对大肠杆菌和金黄色葡萄球菌生物膜的破坏作用较强，能够抑制细菌繁殖(图8A)。然后通过结晶紫染色确定生物膜的生物量，结晶紫染料可以与细菌细胞膜表面的分子和多糖结合。从图8B可以看出，对照组呈深紫色，表明细菌形成了相对完整的生物膜；然而，usAgNPs-PL/CMC水凝胶组形成了几乎透明的薰衣草色，这证明了usAgNPs-PL/ CMC水凝胶可以抑制细菌形成生物膜和生长繁殖。核酸染料PI能与死细菌结合后发出红色荧光，图8C显示对照组表现出微弱的红色荧光，相比之下，usAgNPs-PL/CMC水凝胶组显示较强的红色荧光，说明usAgNPs-PL/CMC水凝胶具有更重要的抗菌能力。基于上述抗菌实验结果，作者尝试探索usAgNPs-PL/CMC水凝胶的抗菌机制，进行了活性氧、丙二醛和ATP水平检测，其中，细菌产生大量活性氧是导致其死亡的主要原因。图8D显示usAgNPs-PL/CMC水凝胶诱导大量活性氧，细胞内活性氧积累的主要后果之一是逐渐建立的氧化应激对膜完整性的损害。因此继续使用丙二醛方法检测细胞脂质过氧化，以确定膜损伤的程度，如图8E显示，usAgNPs-PL/CMC水凝胶组活性氧诱导的丙二醛在大肠杆菌中达到149.94%、在金黄色葡萄球菌中达到127.08%，均高于对照组。为了评估usAgNPs-PL/CMC水凝胶对细菌代谢的影响，测定了细胞内ATP水平，如图8F所示，usAgNPs-PL/CMC水凝胶组中的ATP水平明显低于对照组，表明usAgNPs-PL/CMC水凝胶能够抑制细菌(大肠杆菌和金黄色葡萄球菌)ATP水平。"

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