Fonseca Diana R, Moura Ana, Leiro Victoria, Silva-Carvalho Ricardo, Estevinho Berta N, Seabra Catarina L, Henriques Patrícia C, Lucena Mónica, Teixeira Cátia, Gomes Paula, Parreira Paula, Martins M Cristina L
i3S, Instituto de Investigação e Inovação em Saúde, Rua Alfredo Allen, 208, Porto 4200-135, Portugal; INEB, Instituto de Engenharia Biomédica, Rua Alfredo Allen, 208, Porto 4200-135, Portugal; Faculdade de Engenharia, Departamento de Engenharia Metalúrgica e de Materiais, Universidade do Porto, Rua Dr. Roberto Frias, Porto 4200-465, Portugal.
i3S, Instituto de Investigação e Inovação em Saúde, Rua Alfredo Allen, 208, Porto 4200-135, Portugal; INEB, Instituto de Engenharia Biomédica, Rua Alfredo Allen, 208, Porto 4200-135, Portugal.
Acta Biomater. 2022 Jan 1;137:186-198. doi: 10.1016/j.actbio.2021.09.063. Epub 2021 Oct 8.
MSI-78A (Pexiganan A) is one of the few antimicrobial peptides (AMPs) able to kill Helicobacter pylori, a pathogenic bacterium that colonizes the gastric mucosa of half of the world's population. Antibiotics fail in 20-40% of H. pylori-infected patients, reinforcing the need for alternative treatments. Herein, a bioengineered approach was developed. MSI-78A with a C-terminal cysteine was grafted onto chitosan microspheres (AMP-ChMic) by thiol-maleimide (Michael-addition) chemistry using a long heterobifunctional spacer (NHS-PEG-MAL). Microspheres with ∼4 µm diameter (near H. pylori length) and stable at low pH were produced by spray drying using a chitosan solution with an incomplete genipin crosslinking. A 3 × 10 µg AMP/microsphere grafting was estimated/confirmed by UV/Vis and FTIR spectroscopies. AMP-ChMic were bactericidal against H. pylori J99 (highly pathogenic human strain) at lower concentrations than the free peptide (∼277 µg grafted MSI-78A-SH/mL vs 512 µg free MSI-78A-SH/mL), even after pre-incubation in simulated gastric conditions with pepsin. AMP-ChMic killed H. pylori by membrane destabilization and cytoplasm release in a ratio of ∼10 bacteria/microsphere. This can be attributed to H. pylori attraction to chitosan, facilitating the interaction of grafted AMP with bacterium membrane. Overall, it was demonstrated that the peptide-microsphere conjugation chemistry did not compromise the MSI-78A antimicrobial activity, instead it boosted its bactericidal performance against H. pylori. STATEMENT OF SIGNIFICANCE: Half of the world's population is infected with Helicobacter pylori, a gastric bacterium that is responsible for 90% of non-cardia gastric cancers. Therefore, H. pylori eradication is now advocated in all infected individuals. However, available antibiotic therapies fail in up to 40% patients. Antimicrobial peptides (AMPs) are appealing alternatives to antibiotics, but their high susceptibility in vivo limits their clinical translation. AMP immobilization onto biomaterials surface will overcome this problem. Herein, we demonstrate that immobilization of MSI-78A (one of the few AMPs with activity against H. pylori) onto chitosan microspheres (AMP-ChMic) enhances its anti-H. pylori activity even at acidic pH (gastric settings). These results highlight the strong potential of AMP-ChMic as an antibiotic alternative for H. pylori eradication.
MSI-78A（派昔加南A）是少数能够杀死幽门螺杆菌的抗菌肽之一，这种致病菌寄生于世界上半数人口的胃黏膜中。20%-40%的幽门螺杆菌感染患者使用抗生素治疗无效，这凸显了对替代治疗方法的需求。在此，我们开发了一种生物工程方法。通过硫醇-马来酰亚胺（迈克尔加成）化学反应，使用长链异双功能间隔物（NHS-PEG-MAL），将带有C端半胱氨酸的MSI-78A嫁接到壳聚糖微球（AMP-ChMic）上。通过使用不完全京尼平交联的壳聚糖溶液进行喷雾干燥，制备出直径约4μm（接近幽门螺杆菌长度）且在低pH值下稳定的微球。通过紫外/可见光谱和傅里叶变换红外光谱估计/确认每微球接枝3×10μg AMP。即使在模拟胃环境中与胃蛋白酶预孵育后，AMP-ChMic对幽门螺杆菌J99（高致病性人类菌株）的杀菌浓度也低于游离肽（接枝的MSI-78A-SH约277μg/mL，游离的MSI-78A-SH为512μg/mL）。AMP-ChMic通过膜破坏和细胞质释放以约每微球10个细菌的比例杀死幽门螺杆菌。这可归因于幽门螺杆菌对壳聚糖的吸引力，促进了接枝的AMP与细菌膜的相互作用。总体而言，已证明肽-微球偶联化学不会损害MSI-78A的抗菌活性，反而增强了其对幽门螺杆菌的杀菌性能。重要性声明：世界上半数人口感染幽门螺杆菌，这种胃部细菌导致了90%的非贲门胃癌。因此，目前主张对所有感染者进行幽门螺杆菌根除治疗。然而，现有的抗生素疗法在高达40%的患者中无效。抗菌肽（AMPs）是抗生素的有吸引力的替代品，但其在体内的高敏感性限制了它们的临床应用。将AMP固定在生物材料表面将克服这个问题。在此，我们证明将MSI-78A（少数对幽门螺杆菌有活性的AMP之一）固定在壳聚糖微球（AMP-ChMic）上可增强其抗幽门螺杆菌活性，即使在酸性pH值（胃部环境）下也是如此。这些结果突出了AMP-ChMic作为根除幽门螺杆菌的抗生素替代品的强大潜力。
