(210h) Fusogenic Liposomes Functionalized with Cell Penetrating Peptides for the Treatment of Bacterial Meningitis

Authors: 
Bartomeu Garcia, C., Northeastern University
Webster, T., Northeastern University
Shi, D., Northeastern University

Fusogenic
Liposomes Functionalized with Cell-Penetrating Peptides for the Treatment of
Bacterial Meningitis

text-align:center;line-height:normal"> " times new roman>Caterina Bartomeu Garcia, Di Shi, and Thomas J.
Webster

text-align:center;line-height:normal"> " times new roman>Department of Chemical Engineering, Northeastern
University, Boston, MA, USA

text-align:center;line-height:normal"> " times new roman> 

justify;text-indent:.5in;line-height:normal"> font-family:" times new roman>Bacterial meningitis is one of the most severe
and aggressive infections that leads to permanent disabilities and brain damage
to 20 percent of the population that manage to survive this disease. Bacteria
spreads in the blood stream and reaches the brain site, where it provokes the
inflammation of the meninges and the fluid-filled spaces in-between them. If
not treated in time, bacterial meningitis can lead to permanent brain damage as
well as death. The emergence of drug-resistant bacteria has increased the
difficulty of treatment and the mortality rate. Bacteria with low
susceptibility to current antibiotics lead to the formation of biofilms, which
further compromises the ability of antibiotics to treat this disease, leading
to ineffective treatment and progressed infections. The overuse of antibiotics
not only promotes the proliferation of drug-resistant bacteria, but also
affects other beneficial microbes in our body. To this end, there is a need to develop
a more targeted drug delivery system, able to specifically interact with
bacteria and combat its drug resistance.

justify;text-indent:.5in;line-height:normal"> font-family:" times new roman>Our research is focused on the use of fusogenic
(pH-sensitive) liposomes to promote the destabilization of the bacterial cell
membrane and introduce antibiotics directly inside the bacterial cell cytoplasm.
Previous research reported the ability of these particles to reduce the minimum
inhibitory concentration (MIC) of antibiotic necessary to kill bacteria, as
well as their ability to encapsulate different type of antibiotics. For this
project, our liposome systems were functionalized with a cell-penetrating
peptide TAT (47-57) to increase their permeability through the blood brain
barrier (BBB), one of the main obstacles that modern drugs face for treating
brain diseases.

justify;text-indent:.5in;line-height:normal"> font-family:" times new roman>Liposomes were synthesized by the lipid
film rehydration method and functionalized with TAT (47-57) using wet
chemistry. Liposomes were characterized using transmission electron microscopy
to verify their successful formation and size (approximately 100 nm). They were
loaded with one of three commonly used antibiotics to treat meningitis,
including vancomycin, methicillin, and ampicillin. These were tested against
the three main bacteria that cause this disease, Streptococcus pneumoniae,
methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia
coli
. Growth curve assays, colony forming units, and live/dead fluorescent
staining assays were performed to study the antibacterial properties of our
liposomes against the previously mentioned bacteria. Liposomes were also tested
against astrocytes and endothelial cells, the main cells that form the BBB, in
order to demonstrate their non-cytotoxicity properties. In addition, in
vitro
BBB studies using a static model were used to test the ability of
these particles to cross the BBB. These static models consist of astrocytes
seeded at the bottom of a 24-well plate, with inserts containing endothelial
cells added to each well. After a set incubation time, endothelial cells were
placed inside a separate 24-well plate containing bacteria. Liposomes were
added to study their permeability through the endothelial cell barrier to reach
the bacteria.

justify;text-indent:.5in;line-height:normal"> font-family:" times new roman>Antibacterial results demonstrated significant
antibacterial activity, especially against MRSA with a MIC of 1.7 µg/mL. Cytotoxicity
results showed no interaction of the functionalized and non-functionalized
liposomes with astrocytes and endothelial cells, as well as a significant
reduction of antibiotic cytotoxicity through encapsulation. Initial BBB in
vitro
studies suggested a high permeability of these particles, as the
concentration of bacteria across the endothelial cells was significantly lower
when in the presence of liposomes.

justify;text-indent:.5in;line-height:normal"> font-family:" times new roman>In conclusion, our liposome system demonstrated
significant antibacterial properties, especially towards MRSA and S.
pneumoniae
, while non-cytotoxicity was observed against astrocytes and
endothelial cells. Furthermore, the ability of these particles to cross the BBB
and combat the bacteria was successfully proved with a static BBB model, while also
avoiding the destabilization of the endothelial cell membrane.

justify;text-indent:.5in;line-height:normal"> font-family:" times new roman>Future research is focused on the
interaction of these liposomes with the bacterial cell membrane in order to
better understand the bacterial cell uptake mechanism. Scanning electron
microscopy (SEM) was performed to observe the bacterial cell membrane after
their interaction with the liposome particles (Figure 1). This figure illustrates
the disruption of the bacterial cell membrane with a low concentration of
liposomes, as well as a total disruption of the bacterial cell membrane with
higher concentration of antibiotics. Bacterial cell protein studies will also be
performed, with a focus on the penicillin binding protein 2a, known for causing
bacterial resistance to antibiotics.

justify;text-indent:.5in;line-height:normal"> font-family:" times new roman>This work was supported by funds from
Northeastern University.

.5in;line-height:normal">Figure
1. SEM of (A) MRSA, (B) MRSA with TAT liposomes loaded with 2 µg/mL
methicillin, (C) MRSA with TAT liposomes loaded with 4 µg/mL
methicillin.