(292e) Fusogenic Liposomes Loaded with Antibiotics As a Combined Therapy for Fighting Brain Infections (Award Session)

Meningitis is caused by the inflammation of meninges and the in-between fluid filled spaces. Bacterial meningitis, the most common and the most aggressive form of bacterial infection, is mainly caused by Gram-positive bacterium Streptococcus pneumoniae and Gram-negative bacterium Neisseria meningitidis. Its treatment consists of a combination of antibiotics, that sometimes fail due to their poor permeability through the blood-brain barrier (BBB). Moreover, the emergence of drug-resistant bacteria and their ability to create biofilms have become a global concern, making it increasingly difficult to treat these infections. When bacterial biofilms are formed, the ability of antibiotics to treat these diseases is diminished, leading to ineffective treatment. In severe cases, meningitis can lead to death. Therefore, there is a need to create alternative treatments for bacterial infections of brain, such as bacterial meningitis.

The use of nanotechnology in medicine, especially in drug delivery, is one of the most promising strategies to solve the problem of antibiotic resistance. Here, we report the use of fusogenic (pH-sensitive) liposomes, as drug delivery systems to target the bacteria cell membrane. These liposomes release the antibiotic directly at the site of infection, notably inside the bacterial cell. pH-sensitive liposomes have been previously used to target bacteria due to their ability to interact with the phospholipids present in the bacteria outer membrane, destabilizing it. For the purpose of this study, liposomes were functionalized with a cell-penetrating peptide, TAT (47-57) to increase their permeability through the blood-brain barrier, which is the primary challenge in treating meningitis.

Lipid film rehydration method was used to synthesize the liposomes, followed by their functionalization with TAT (47-57) peptide using wet chemistry. Liposomes were characterized by transmission electron microscopy (TEM) to verify their formation, and size of about 100 nm. These liposomes were loaded with three different antibiotics, methicillin, ampicillin and methicillin, with a loading efficiency of around 68%, 72% and 83% respectively.

Bacterial growth curve studies, and colony forming studies results showed that TAT functionalized liposomes loaded with vancomycin and methicillin were successfully able to kill Streptococcus pneumoniae, and methicillin-resistant Staphylococcus aureus respectively. These results not only demonstrate the ability of these particles to decrease the minimum inhibitory concentration (MIC) of antibiotic necessary to kill bacteria, but also their ability to combat drug-resistant bacteria.

In vitro cytotoxicity test using fusogenic liposomes against astrocytes and endothelial cells, primary cells that form the BBB, showed no interaction between our nanoparticles and both cell lines, as well as a significant reduction of antibiotic cytotoxicity through encapsulation.

A novel static in vitro BBB model was used to determine the ability of these nanoparticles to cross to blood brain barrier. This model consisted 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 on the other side of the membrane. Initial studies with these static models showed the ability of our functionalized nanoparticles to not just cross the blood brain barrier, but also significantly decrease the bacteria population on the other site.

Future studies will be focused on a better understanding of the interaction mechanism between our TAT functionalized liposomes and bacterial cells, and on the use of dynamic and more sophisticated BBB models to better mimic the liposome permeability through the BBB.

This work was supported by Department of Chemical Engineering, Northeastern University.