Hypoxia-sensitive adjuvant loaded liposomes enhance the antimicrobial activity of azithromycin via phospholipase-triggered releasing for Pseudomonas aeruginosa biofilms eradication.

Robust biofilms and the complex airway environment with thick sputum, local hypoxia and persistent inflammation induce the intractability of chronic pulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). Herein, we proposed a type of antibiotic-adjuvant liposomes (NANO@PS-LPs), co-incorporating azithromycin (AZI), adjuvant (2-nitroimidazole derivative, 6-NIH) and biofilm dispersant (nitric oxide donor, DETA NONOate). NANO@PS-LPs possessing negatively-charged surface and good hydrophilicity could easily penetrate through the sputum layer, then disassembled triggered by overexpressed phospholipase A2 (PLA2) in the microenvironment around biofilms. Nitric oxide produced by DETA NONOate promoted P. aeruginosa biofilms dispersal. 6-NIH was reduced to 2-aminomidazole derivative (6-AIH) under a hypoxic condition, and hence acted as an AZI adjuvant to enhance the antibacterial activity of AZI. It was found that NANO@PS-LPs could significantly eliminate mature P. aeruginosa biofilms, effectively kill dispersed bacteria, inhibit the metabolism of survivors and prevent P. aeruginosa adherence to airway epithelial cells, accordingly restrain recurrent infections. Additionally, NANO@PS-LPs performed a remarkable advantage in killing AZI-resistant P. aeruginosa and removing their biofilms. In summary, NANO@PS-LPs present a potential nano-strategy to treat stubborn pseudomonal pulmonary infections and overcome correlative drug resistance.

[Biofilm Eradication Four-Step Strategy: Study of Using Self-Assembled Azithromycin/Rhamnolipid Nanoparticles for Removing Pseudomonas aeruginosa Biofilm].

OBJECTIVE: To investigate the in vitro eradicative effect of self-assembled azithromycin/rhamnolipid nanoparticles (AZI-RHL NPs) on P seudomonas aeruginosa ( P. aeruginosa) biofilm. METHODS: AZI-RHL NPs were prepared and characterized. The minimum inhibitory concentration (MIC) of AZI-RHL NPs on planktonic P. aeruginosa was measured by the broth microdilution method. The eradicative effect of AZI-RHL NPs on P. aeruginosa biofilm was evaluated via crystal violet staining and SYTO 9/PI live/dead staining. Fluorescence labeling was used to measure the eradicative effect of NPs on extracellular polymeric substances (EPS). In addition, crystal violet staining was performed to evaluate the inhibitory effect of AZI-RHL NPs on the adhesion of P. aeruginosa on human bronchial epithelial BEAS-2B cells. To investigate the ability of AZI-RHL NPs to penetrate mucus, the interaction between NPs and mucin was measured via particle size changes after co-incubation with mucin solution. RESULTS: The AZI-RHL NPs had a particle size of about 121 nm and were negatively charged on the surface, displaying a high encapsulation efficiency and a high drug loading capacity of 96.72% and 45.08% for AZI, respectively and 99.38% and 53.07% for RHL, respectively. The MIC of AZI-RHL NPs on planktonic P. aeruginosa was half of that of using AZI alone. AZI-RHL NPs displayed the capacity to effectively destroy the biofilm structure and remove the proteins and polysaccharides in EPS, eradicating biofilms in addition to reducing the survival rate of bacteria in the biofilm. AZI-RHL NPs were shown to have inhibited P. aeruginosa adhesion on BEAS-2B cells and prevented the residual bacteria from forming a new biofilm. There was no significant change in the particle size of NPs after co-incubation with mucin solution, indicating a weak interaction between NPs and mucin, and suggesting that NPs could penetrate the mucus and reach the P. aeruginosa infection sites. CONCLUSION: AZI-RHL NPs were able to effectively enhance the removal of P. aeruginosa biofilm through a four-step strategy of biofilm eradication, including penetrating the mucus, disintegrating the biofilm structure, killing the bacteria dispersed from biofilm, and preventing the adhesion of residual bacteria. We hope that this study will provide a replicable common strategy for the treatment of refractory infections caused by P. aeruginosa and other types of biofilms.

Self-assembled nanomedicine combining a berberine derivative and doxorubicin for enhanced antitumor and antimetastatic efficacy via mitochondrial pathways.

Mitochondria play a central role in cancer progression and tumor metastasis, and nanomedicines targeting mitochondria have emerged as a promising strategy for tumor therapy. However, mitochondria targeting strategies have not been widely explored in the inhibition of tumor metastasis, and they have disadvantages of complicated preparation, low drug loading, systemic toxicity of the carriers and poor accumulation at tumor sites. Here we firstly developed self-assembled nanodrugs with a high drug loading (∼68%) comprised of a berberine derivative (Ber) and doxorubicin (Dox) by a simple nano-precipitation method, which successfully altered the target location of Dox from the nucleus to mitochondria and therefore inhibited the proliferation, invasion and migration of MDA-MB-231 cells by triggering cell apoptosis. The surface of nanodrugs was modified with DSPE-PEG-folic acid (DSPE-PEG-FA) and hyaluronic acid (HA) for precise tumor recognition and enhanced accumulation (HA-FA-BD NDs). Upon arrival at the tumor site with the help of the enhanced permeability and retention (EPR) effect, the partial degradation of HA by hyaluronidase (HAase) at the tumor site allowed the partial exposure of the positively charged FA-BD NDs to the cells, then nanodrugs would accumulate and enter tumor cells by dual binding to both folic acid (FA) and CD-44 receptors. Once internalized into lysosomes, both the HA outer shell and DSPE-PEG-FA of nanodrugs were degraded or decomposed completely to expose positively charged BD NDs. Driven by delocalized lipophilic cations, nanodrugs could escape from lysosomes and reach mitochondria to induce a cascade reaction and finally cell apoptosis, as well as suppressing matrix metalloprotease (MMP)-2 and -9 activities and finally cell migration and invasion. In a xenograft mice model of MDA-MB-231 breast cancer cells, the nanodrugs repaired the defects in Mfn 1/Drp 1 mitochondrial proteins, suppressed the activity of MMP-2 and -9, and significantly inhibited tumor cell proliferation and pulmonary metastasis. Our study showed a promising strategy for the treatment of metastatic breast cancer by targeting mitochondria followed by enhanced apoptosis.

Antibacterial self-assembled nanodrugs composed of berberine derivatives and rhamnolipids against Helicobacter pylori.

The prevalence of infections with Helicobacter pylori (H. pylori) has progressively increased worldwide, which demonstrated to be closely correlated to its biofilm formation. H. pylori biofilms protect the bacteria by significantly decreasing their sensitivity to antibiotics. Moreover, H. pylori colonizes on the gastrointestinal tract epithelium which is covered by mucus layer, acting as another barrier to prevent antibacterial agents from reaching the colonization sites. Herein, we prepared four types of versatile self-assembled nanodrugs (BD/RHL NDs) containing lipophilic alkyl berberine derivatives (BDs) and rhamnolipids (RHL) to overcome the dual obstructions of both mucus layer and biofilms. Molecular dynamics simulations estimated that the driving forces for self-assembly of BD/RHL NDs were electrostatic and hydrophobic interactions. BD/RHL NDs, characterized by appropriate size, negative charge and enhanced hydrophilicity, successfully penetrated through mucus layer without interacting with mucins. In in vitro experiments, BD/RHL NDs exhibited substantial ability to eradicate H. pylori biofilms by destroying their extracellular polymeric substances (EPS) and killing planktonic H. pylori. Furthermore, BD/RHL NDs inhibited the adherence of H. pylori on both biotic and abiotic surfaces, therefore cut off the critical step of the biofilm re-formation which was associated with the recrudescence of infections. In an H. pylori-infected mice model, C10-BD/RHL NDs group showed 40 folds less remnant H. pylori and greater mucosal protection compared with the conventional clinical triple therapy. In conclusion, BD/RHL NDs could penetrate through mucus layer and effectively eradicate H. pylori biofilms in vitro and in vivo, providing a novel strategy for clinical treatment of biofilm-related infections.