Therapeutic Delivery of Antibodies Across the Blood-Brain Barrier for Neurologic Rabies

Saturday, October 26, 2024

1:35 PM - 1:40 PM EST

Location: Room 342

Presenting Author(s)

Pablo Lozano

Undergraduate Student
Clemson University
Simpsonville, South Carolina, United States

Primary Investigator(s)

Jessica Larsen, PhD

Carol and John Cromer '63 Family Endowed Associate Professor
Clemson University Department of Chemical and Biomolecular Engineering, South Carolina, United States

Introduction: Rabies is a viral infection normally associated with small mammals like dogs and bats. This number has been controlled with vaccination campaigns in dogs as well as case tracking which helps control outbreaks, but there still a therapeutic gap when we move away from prevention into treatment of a rabies infection. Rabies is neurological in nature, which means the virus attacks the central nervous system of the host organisms. This makes therapeutics challenging since they need to cross the blood-brain barrier (BBB). Additionally, this therapeutic gap can affects humans as rabies is a zoonotic virus which leads to around 59,000 human deaths per year in the world. We aim to address the therapeutic gap by developing polymersomes capable of crossing the BBB, targeting the central nervous system, and depositing a payload of monoclonal antibodies.

Polymersomes are self-assembling vesicles made from block copolymers with both hydrophobic and hydrophilic portions. This makes polymersomes amphiphilic nanoparticles that can be loaded with different molecules, similar to biological vesicles. These polymersomes are used extensively in the Larsen Lab for transport of biologics to difficult to reach tissues, like the brain or the nerves, through non-invasive administration. We hypothesize that we can develop polymersomes to encapsulate therapeutic, monoclonal antibodies to treat neurological rabies in canine models effectively, thus paving the way for further research on neurological therapeutics with potential human applications.

Materials and

Methods: The Center of Nanotherapeutic Strategies in the Central Nervous System (or the Larsen Lab) conducted preliminary studies on optimizing the polymersome structure and the addition of targeting ligands for delivery across the BBB. We used poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) to create our polymersomes through a solvent infection method. The polymersomes were characterized using the Malvern Nanozetasizer. We attached targeting ligand Apolipoprotein E (ApoE) to polymersomes using the same solvent injection protocol with the addition of ApoE protein into the aqueous solvent. These samples were also characterized for protein adhesion using a Lowry assay.

PEG-b-PLA polymersomes were loaded with a model fluorescent antibody (AF488 goat anti-mouse IgG) via dialysis to calculate loading efficiency. Following successful encapsulation into the polymersomes, release studies in pH 7.4 (physiologic) and pH 5.5 (release conditions) were performed to confirm stimuli-responsive release.

Results, Conclusions, and Discussions: PEG-b-PLA polymersomes were synthesized as both untagged and tagged. The untagged samples had a diameter of 109.1 ± 2.71 nm, a stable surface charge of -22.9 ± 0.702 mV, and a uniform polydispersity index of 0.115 ± 0.024. Tagged samples are expected to increase in diameter and polydispersity index due to the protein adhesion on the surface of the polymersomes. Additionally, the surface charge is expected to be like their untagged counterpart; ApoE-tagged polymersomes had a diameter of 159.1 ± 5.44 nm, a stable surface charge of -24.9 ± 2.03 mV, and a uniform polydispersity index of 0.281 ± 0.071.

Additionally, ApoE-tagged polymersomes were tested to see the ratio of protein adhesion when compared to untagged polymersomes. All tagged polymersomes showed protein adhesion ratios higher than 1 with an average of 17.7 times more protein adhesion after the addition of ApoE. With these promising characterization results, it was determined encapsulation studies could performed on both untagged and tagged polymersomes samples.

The untagged polymersomes had an 75.4 ± 6.8% encapsulation efficiency which meant 7.54E-4 ± 6.8E-5 milligrams of the model antibody per milligram of polymersomes were encapsulated. The tagged polymersomes had a 56.2 ± 26.1% encapsulation efficiency which meant 5.62E-4 ± 2.6E-4 milligrams of the model antibody per milligram of polymersomes were encapsulated. This decrease is expected as the tagged polymersomes have a layer of protein adhesion on the surface of the polymersomes which will hinder the encapsulation of therapeutics.

Release studies confirmed antibodies are released more rapidly in an acidic environment, as expected due to the polyester PLA backbone of the polymersomes.

In summary, we can synthesize polymersomes with appropriate sizes, stable surface charges, and monodisperse distributions both with and without a targeting ligand. Additionally, we can encapsulate an antibody payload and release it in an acidic environment. Given this, we proceeding to the study of a specific rabies antibody. This would allow us to synthesize a therapeutic option for neurological rabies in canine models, but this is would just be the superficial benefit of the project. This will pave the way for neurological therapeutics as a growing research field.

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