(498c) Liposomes Functionalized with Cell-Penetrating Peptides As a Novel Treatment for Bacterial Meningitis

Liposomes Functionalized with Cell-Penetrating
Peptides as a Novel Treatment for Bacterial Meningitis

Caterina Bartomeu, Di Shi, and Thomas J. Webster

Department of Chemical Engineering, Northeastern
University, Boston, MA, USA

Bacterial meningitis is a very
serious infection that can lead to permanent disabilities and death in as
little as a few hours if left untreated. It is a brain inflammatory disease caused
by inflammation of the meninges and in-between fluid-filled spaces. Severe cases
can lead to permanent brain damage as well as death. Brain inflammatory
diseases have become a global concern in clinical care due to the recent
emergence of antibiotic-resistant bacteria, making it increasingly difficult to
treat these infections. By forming a complex polysaccharide matrix known as biofilms,
bacteria protect themselves from environmental and external stimuli such as
antibiotics, allowing them to spread their antibiotic resistance within the
bacterial community. The current broad-spectrum antibiotics face great
difficulties in penetrating these biofilms, leading to ineffective treatment
and progressed infection. In addition, the overuse of antibiotics promotes
resistance in bacteria and leads to longer stays in hospitals and poorer
patient care outcomes. Therefore, there is a need to develop alternative
treatments for brain infection diseases such as bacterial meningitis. 

To address this need, we report here
the use of pH-sensitive liposomes as drug carriers to deliver targeted
antibiotics at the site of infection. The ability of pH-sensitive liposomes to
fuse with the bacterial cell membrane has been reported previously,¹ as well as the successfully encapsulation of
different particles, such as antibiotics and metal nanoparticles, inside their
vesicles or in-between their phospholipid bilayers.² Liposomes were
functionalized with a cell-penetrating peptide, TAT (47-57) in order to be able
to cross the blood-brain barrier (BBB), which is one of the main challenges for
treating brain diseases.³

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. Bacterial growth curve studies were performed to
evaluate the antibacterial effect of the liposomes. Also, the cytotoxicity of
the liposomes was tested with astrocytes and endothelial cells. In addition to these studies, we also performed BBB
studies using an in vitro model where
astrocytes were seeded at the bottom of a 24 well plate, then inserts with
endothelial cells were added to each well. After five days, the endothelial
cell inserts were placed inside a separate 24 well plate containing bacteria. TAT-functionalized
liposomes were added to see if they were able to cross the BBB and fight the
bacteria located at the bottom of the plate.

Initial experiments were focused on
the antibacterial properties of the TAT-functionalized liposomes. Their inhibitory
effect on bacterial growth was tested with bacteria commonly associated with
bacterial meningitis, including Streptococcus
pneumoniae, methicillin-resistant
Staphylococcus aureus (MRSA), and Escherichia
coli. The results from these antibacterial studies demonstrated excellent
growth inhibitory effects of TAT-functionalized liposomes loaded with
methicillin for treating MRSA.

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.

Initial BBB in
vitro studies showed that the readings of the transendothelial
electrical resistance (TEER) decreased when the endothelial cells inserts were
added to the 24 well plate with bacteria (Figure 1), indicating that the
endothelial cells were dying. However, this decrease in TEER was notably less
with the inserts containing our TAT-functionalized liposomes, and after the
first hour the TEER values increased. This seems to indicate that our particles
were able to cross the BBB and kill the bacteria located on the other side.  

Figure 1. Blood-brain
barrier static assay. TEER values obtained with TAT-functionalized liposomes
loaded with methicillin (TAT-LipoMet) to fight MRSA

All results obtained for TAT-functionalized liposomes,
including their low minimum inhibitory concentration (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. Next
steps in this research include additional BBB studies, utilizing static and
dynamic BBB models, and investigating the effect of the liposome treatment on
the genetic properties of the bacterial cell membrane, to better understand
their cellular uptake mechanism.

This work was supported by funds from Northeastern
University.

1. Pignatello,
R., et al., Science Against Microbial Pathogens, 1,
52-60, 2011

2. Martinez-Gonzalez,
R., et al. International Journal of
Molecular Science Int. 17(8), 1209, 2016

3. Stalmans,
S., et al., PLoS ONE, 10, pp. 1-22, 2015