Title：Synchronous Sterilization and Immunoreaction Termination for Corneal Transparency Protection in Treating Pseudomonas aeruginosa Induced Bacterial Keratitis

Journal: Advanced Materials

IF: 27.4

Original link：https://advanced.onlinelibrary.wiley.com/doi/full/10.1002/adma.202419209

Reporter：Liang Qiu-23-master

In the treatment of infectious keratitis, therapeutic strategies often prioritize enhancing bactericidal efficacy. However, endotoxins released from Gram-negative bacteria cause inflammatory reaction, leading to corneal structural damage and scar formation. Given that polymyxin B (PMB) can bind and neutralize lipopolysaccharide (LPS), this study employs large-pore mesoporous silica nanoparticles (lMSNs) grafted with PMB as carriers for cationic antibacterial carbon quantum dots (CQDs) to prepare CQD@lMSN-PMB, which enables synchronous sterilization and endotoxin neutralization. In the acidic infectious microenvironment, the accelerated release of CQDs eliminates 99.88% bacteria within 2 h, effectively substituting immune mediated sterilization. Notably, CQD@lMSN-PMB exhibits exceptional LPS neutralization performance (2.22 µg LPS/mg CQD@lMSN-PMB) due to its high specific surface area. In an infectious keratitis model, inflammation subsides significantly within the first day of CQD@lMSN-PMB intervention and is completely resolved by day 3. By day 2, interleukin-1β, interleukin-6 and tumor necrosis factor-α in CQD@lMSN-PMB group decrease by 86.99%, 91.15%, and 77.56%, respectively, compared to the CQDs-only sterilization group. Ultimately, corneal integrity and transparency are preserved, with suppressed expressions of fibrosis-related factors including matrix metalloproteinase 9, transforming growth factor-β and α-smooth muscle actin. Therefore, this synchronous sterilization and endotoxin neutralization strategy outperforms monotherapy strategies focused solely on sterilization or endotoxin neutralization.

CQD@lMSN-PMB achieves efficient sterilization by releasing spermidine CQDs, and uses PMB covalently grafted in the lMSNs pores to neutralize endotoxins through electrostatic and hydrophobic interactions, thereby effectively inhibiting the inflammatory response triggered by LPS. In the corneal inflammation model caused by Pseudomonas aeruginosa infection, the levels of pro-inflammatory cytokines (such as IL-1β, IL-6, and TNF-α) in the CQD@lMSN-PMB treatment group showed a downward trend after the intervention began, without reaching an inflammatory peak. The corneal prognosis was good, and no obvious scarring was observed. CQD@lMSN-PMB successfully blocked the progression of the inflammatory response through efficient sterilization and endotoxin neutralization, thereby effectively protecting the structural integrity and transparency of the cornea.

1. Fabrication and Characterization of CQD@IMSN-PMB

The figure introduces the preparation and screening of CQD@lMSN-PMB. Three different pore-sized MSN-PMBs were synthesized, namely small-pore sMSN-PMB, medium-pore mMSN-PMB and large-pore lMSN-PMB, with pore diameters of 2.51, 7.48 and 11.66 nm, respectively. In vitro LPS adsorption experiments indicated that lMSN-PMB had the best LPS adsorption performance, and was further loaded with antibacterial CQDs. CQDs can be rapidly released in the acidic infection microenvironment. Thanks to the large specific surface area of MSNs, CQD@lMSN-PMB has an efficient LPS adsorption capacity of approximately 2.22 μg LPS/mg CQD@lMSN-PMB.

2. Bactericidal Ability of CQD@lMSN-PMB

The figure shows the sterilization performance and antibacterial mechanism of CQD@lMSN-PMB. The antibacterial experiments prove that CQD@lMSN-PMB has an efficient sterilization ability. It can kill 99.88% and 99.99% of Pseudomonas aeruginosa respectively within 2 hours and 4 hours. CQD@lMSN-PMB achieves efficient sterilization by destroying the bacterial cell membrane and affecting the permeability of the bacterial membrane.

3.  Endotoxin Neutralization and Anti-Inflammatory Effect of CQD@lMSN-PMB

The figure shows the effect of CQD@lMSN-PMB in neutralizing and inhibiting oxidative stress and inflammatory responses through LPS. The experimental results indicate that CQD@lMSN-PMB reduces the generation of ROS and NO, blocks the TLR4 signaling pathway, lowers the expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), significantly inhibits the oxidative stress and inflammatory responses of macrophages, and blocks the activation of the inflammatory response from the source by neutralizing LPS.

4. In Vivo Biocompatibility and Therapeutic Effect of P. aeruginosa-Infected Keratitis

The figure analyzes the gene expression and signaling pathways affected by CQD@lMSN-PMB. The results show that the genes downregulated by CQD@lMSN-PMB are closely related to the genes highly expressed in response to LPS stimulation, suggesting that CQD@lMSN-PMB mainly inhibits inflammation within macrophages by neutralizing LPS. CQD@lMSN-PMB mainly terminates the subsequent inflammatory cascade reaction by inhibiting the activation of the NF-κB signaling pathway.

5. In Vivo Therapeutic Efficacy of CQD@lMSN-PMB

The figure shows the therapeutic effect of CQD@IMSN-PMB in Pseudomonas aeruginosa keratitis. The results indicate that the inflammatory response of the cornea in the CQD@IMSN-PMB group was rapidly controlled, the corneal edema was significantly reduced, the ocular surface bacteria were rapidly cleared, and the corneal transparency was effectively restored.

6. In Vivo Anti-Inflammatory Effect of CQD@lMSN-PMB

The figure shows the anti-inflammatory effect of CQD@lMSN-PMB in Pseudomonas aeruginosa keratitis. The results indicate that at the early stage of intervention, CQD@lMSN-PMB can inhibit the rising trend of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), prevent the formation of peak levels of inflammatory factors, and significantly reduce the level of corneal inflammation.

7. In Vivo Corneal Healing Assessment of CQD@lMSN-PMB

The figure shows the promoting effect of CQD@lMSN-PMB on corneal healing in Pseudomonas aeruginosa keratitis. The results indicate that the corneal prognosis of the CQD@lMSN-PMB treatment group was good, with intact structure, clear layers, decreased expression of fibrosis-related factors, and no significant formation of corneal scarring.

Conventional infectious keratitis treatments often fail to simultaneously control inflammatory damage, resulting in scar formation and reduced corneal transparency. In this study, a novel nanomaterial, CQD@lMSN-PMB, was designed to achieve potent bacterial eradication while synchronously neutralizing endotoxins released by lysed Gram-negative bacteria. The cationic bactericide CQDs were rapidly released, effectively replacing the immune system's bactericidal function. More importantly, lMSNs covalently grafted with PMB exhibited a high-affinity endotoxin-scavenging capability, leveraging their extensive specific surface area to effectively halt inflammatory responses. In the P. aeruginosa-infected keratitis model, proinflammatory cytokine levels (IL-1β, IL-6, TNF-α) declined sharply from the first day of intervention without a discernible inflammatory peak. Notably, only in the CQD@lMSN-PMB group did the cornea maintain structural integrity and transparency, with no significant epithelial defects, ECM degradation, or scar formation. This therapeutic approach demonstrates a significant advantage over strategies based solely on either bacterial elimination or endotoxin neutralization, offering a promising reference for managing other Gram-negative bacterial infection-related inflammatory diseases.