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Considering that some antibacterial agents can identify the outer structure of pathogens like cell wall and/or cell membrane, we explored a self-enhanced targeted delivery strategy by which a small amount of the antibiotic molecules were modified on the surface of carriers as targeting ligands of certain bacteria while more antibiotic molecules were loaded inside the carriers, and thus has the potential to improve the drug concentration at the infection site, enhance efficacy and reduce potential toxicity. In this study, a novel targeted delivery system against methicillin-resistant Staphylococcus aureus (MRSA) pneumonia was constructed with daptomycin, a lipopeptide antibiotic, which can bind to the cell wall of S. aureus via its hydrophobic tail. Daptomycin was conjugated with N-hydroxysuccinimidyl-polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine to synthesize a targeting compound (Dapt-PEG-DSPE) which could be anchored on the surface of liposomes, while additional daptomycin molecules were encapsulated inside the liposomes. These daptomycin-modified, daptomycin-loaded liposomes (DPD-L[D]) showed specific binding to MRSA as detected by flow cytometry and good targeting capabilities in vivo to MRSA-infected lungs in a pneumonia model. DPD-L[D] exhibited more favorable antibacterial efficacy against MRSA than conventional PEGylated liposomal daptomycin both in vitro and in vivo. Our study demonstrates that daptomycin-modified liposomes can enhance MRSA-targeted delivery of encapsulated antibiotic, suggesting a novel drug delivery approach for existing antimicrobial agents.
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Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) or high glucose on the toll-like receptor 4 (TLR4) expression,inflammatory cytokines and fibrotic factors in human tubular epithelial cells (HK-2),revealing the innate immune-related pathogenesis of diabetic nephropathy (DN) which may have clinical implications.Methods Three TLR4 siRNA sequences were designed and synthetized.After transfection,the most effective siRNA was selected to use for further expriments.The experiment consisted of 2 parts.Part 1:Cells were divided into three groups:normal-glucose group (NG,5.5mmol/L glucose),mannose group (M,5.5 mmol/L glucose + 19.5 mmol/L mannose),High-glucose group (HG,25 mmol/L glucose),preliminary validated the effects of high glucose and high osmotic pressure.Part 2:Cells were divided into seven groups:NG group,HG group,Ang Ⅱ group,Ang Ⅱ + negative group,HG+ negative group,Ang Ⅱ + siRNA group and HG+ siRNA group.Real time PCR was used to analyze the mRNA expression of TLR4,myeloid differentiation factor 88 (MyD88),heat shock protein 47 (HSP47).Western blotting was used to observe the protein expression of TLR4,MyD88,HSP47,NF-κB,type Ⅳ collagen (ColⅣ).ELISA was used to detect the expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6).Results Compared with NG group,TLR4,MyD88,HSP47 mRNA and TLR4,MyD88,NF-κB,ColⅣ,HSP47 protein were highly expressed under high glucose or Ang Ⅱconditions (P ＜ 0.01),and the expression levels of MCP-1 and IL-6 also increased significantly (P ＜ 0.01).Compared with HG or Ang Ⅱ group,the above indicators were obviously inhibited in the TLR4 siRNA groups (P＜0.01).Comparison between blank vector transfected groups and HG group as well as Ang Ⅱ group indicated no statistic significance (P ＞ 0.05).Conclusions Both Ang Ⅱ and high glucose stimulate TLR4 expression,which result in the up-regulation of inflammatory and fibrotic factors in HK-2.Specific target of TLR4 gene silencing can block the TLR4 pathway that is activated by high glucose and Ang Ⅱ,and thus reduce the inflammatory and fibtogenic factors' release.TLR4 signal is the common innate immune response pathway which induces the release of inflammatory and fibrotic factors in HK-2 under high glucose or high angiotension conditions.