(633e) Chemical Modifications to Polysaccharide-Based Microparticles Enabling Efficient Conjugation of Protein Antigens

Gallovic, M. D., Northwestern University
Bandyopadhyay, S., The Ohio State University
Collier, M. A., The Ohio State University
Wyslouzil, B. E., The Ohio State University
Bachelder, E. M., University of California, Berkeley
Ainslie, K. M., University of North Carolina - Chapel Hill

Drug delivery systems using biodegradable polyesters such as poly(lactic-co-glycolic acid) (PLGA) continue to be a popular strategy for achieving sustained protein antigen delivery in subunit vaccine formulations.  Unlike these acid insensitive polyesters, acid-degradable polymers can exploit the acidic environment (pH 4.5 – 6.5) within phagolysosomal compartments of antigen presenting cells.  The two primary types of acid-degradable polymers are those with acid-sensitive backbones (e.g. poly(β-amino esters)) or those that contain acid-cleavable pendant side groups (e.g. acetal derivatized dextrans, Ace-DEX).  Both types of polymers are promising candidates for sustained antigen delivery, but Ace-DEX polymers are specifically attractive due to their acid-sensitivity, pH-neutral degradation products, tunable hydrolytic degradability, and cytocompatibility. 

Bulk antigen encapsulation in polymer delivery vehicles such as Ace-DEX microparticles (MPs) can be accomplished by commonly used techniques such as emulsification/solvent evaporation, which expose the antigen to denaturing conditions.  Alternatively, the conjugation of antigen to the surface of preformed MPs is a more benign approach that avoids exposure to harsh particle fabrication steps.  Furthermore, as opposed to bulk encapsulation, antigens displayed on the surface of polymer MPs may result in an enhanced immune response.

Protein antigens can be immobilized on the surface of preformed particles by covalent and noncovalent (e.g. adsorption) interactions.  The flexibility of Ace-DEX’s glucose monomer offers multiple options for synthetic modifications and covalent antigen conjugation.  Among various covalent bond-forming reactions, the widely used “click conjugation” of one protein molecule to a single polymer end-group may show inefficient protein delivery.  As the biodegradable polymer chain begins to degrade there is a high probability of premature release of these single end-group bound proteins.  We envision that conjugating linkers along the polymer backbone instead of just the end will result in a high degree of surface functionalization and more sustained antigen delivery.  In this work, we report three novel synthetic strategies to covalently conjugate an antigen to preformed chemically-modified Ace-DEX MPs by both hydrolytically reversible and irreversible linkages generated along the full length of Ace-DEX’s backbone.

The first two schemes, which feature Ace-DEX MPs modified with either an acid-labile or a hydrolytically irreversible linkage, require an antigen chemically-modified with a maleimide group.  Although maleimide modification kits are commercially available and widely used, the development of a synthetic strategy to conjugate unmodified antigen to the surface of a functionalized Ace-[O]-DEX particle would simplify the conjugation scheme, avoid any undesirable protein modification conditions, and significantly broaden the applications of protein-particle conjugates.  We present a third synthetic route to obtain a functionalized Ace-DEX polymer and irreversibly conjugate an unmodified antigen on the Ace-DEX MP surface.

Using a model ovalbumin antigen, we have achieved promising protein conjugation efficiencies using each of these schemes.  Ongoing in vivo vaccine studies utilizing a toll-like receptor agonist as an adjuvant are being used to compare each of these novel strategies to a bulk encapsulated antigen.  We believe these novel strategies have encouraging applications for subunit antigen delivery systems.