(525c) TAT-Functionalized Liposomes for the Treatment of Meningitis

Brain inflammatory diseases such as bacterial meningitis have recently become a global concern in clinical care due to the recent emergence of antibiotic-resistant bacteria, making it increasingly difficult to treat these infections. Treatment is made further difficult once these bacteria form a biofilm, a complex polysaccharide matrix that both protects the bacteria from environmental stimuli and allows for the spread of antibiotic resistance within the bacterial community. Broad-spectrum antibiotics, which are commonly used to treat such infections, face difficulties penetrating these biofilms and the outer bacterial cell wall. More importantly, the overuse of broad-spectrum antibiotics in clinical care is prevalent and often ineffective, leading to increased drug resistance in bacteria and poorer patient care outcomes. Therefore, there is a growing need to develop an alternative means for treating bacterial meningitis that do not rely on the use of broad-spectrum antibiotics.

To address this need, we report the use of fusogenic liposomes to deliver targeted antibiotics at the site of infection, notably inside the bacterial cell using cell penetrating peptides (TAT 47-57). Liposomes have recently been demonstrated as effective drug carriers, though combining this approach with TAT peptides to deliver drugs inside bacteria as a way to combat bacterial meningitis which has yet to be fully explored and would be a novel means of treatment. To this end, we investigated the effect of TAT-functionalized liposomes to inhibit the growth of bacteria commonly associated with bacterial meningitis, including Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli.

Liposomes were prepared by lipid film rehydration, functionalized with TAT (47-57) using wet chemistry, and characterized using transmission electron microscopy to both determine spherical diameter (~100 nm) and verify successful liposome formation. Liposomes were loaded with one of three commonly used antibiotics to treat these bacterial infections, including vancomycin, methicillin, and ampicillin. The inhibitory effect on bacterial growth of the liposomes was determined using bacterial growth curves and determining bacteria colony forming units, and this system was compared against non-TAT-functionalized liposomes and against free antibiotic.

The results from this study demonstrated excellent growth inhibitory effects of TAT-functionalized liposomes loaded with methicillin for treating MRSA, with a reported minimum inhibitory concentration (MIC) of 1.7 µg/mL, well below the needed dosage for free antibiotic (5 µg/mL). A similar decrease in bacteria population was also demonstrated using TAT-functionalized liposomes loaded with vancomycin to treat S. pneumoniae (MIC of 1 µg/mL). The TAT-functionalized liposomes presented the highest antibacterial activity in all assays performed, followed by non-functionalized liposomes, and lastly free antibiotics. Cytotoxicity assays with endothelial cells and astrocytes demonstrated a notable increase in percent cell viability using functionalized and non-functionalized liposomes in comparison with free antibiotics. Results obtained with a methicillin concentration of 5 µg/mL showed only 20% cell viability for the free antibiotic, while for liposomes it was significantly higher at 90%.

All results obtained for TAT-functionalized liposomes, including their low MIC with respect to the use of free antibiotics plus their remarkable high percentage of cell viability with astrocytes and endothelial cells, provides evidence that this liposomal system can be a safe, alternative means for treating bacterial meningitis.

This work was supported by funds from Northeastern University.