(723e) Altering PLGA-Peg, PLGA and Peg Oligomer Extension to Understand Driving Forces behind Protein/Polymer Binding, Using Atomistic Molecular Dynamics.

Introduction: Protein therapeutics have the potential to treat a wide range of life-threatening diseases and to alleviate crippling symptoms in affected patient populations. When compared to small molecule drugs, proteins can provide high specificity and complexity in their function, have little effect on normal biological processes and can be used to treat a wide array of diseases. Despite these advantages, protein drugs delivered in free form can cause adverse effects, including protease recognition and cleavage once in the bloodstream, development of cross-reactive neutralizing antibodies, and an increased risk of injection-site or viral infections. Polymer nanoparticles can encapsulate proteins and improve protein delivery and biocompatibility by providing protection against degradation and immune recognition, controlled release, assistance in crossing biological barriers, and targeting specific sites of action. Nanoparticles comprised of poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) are non-toxic and biodegradable. Using this diblock copolymer, desirable nanoparticle characteristics, such as neutral surface charge and uniformity in size and dispersity, can be achieved but only after extensive manipulation of formulation parameters. Specifically, finding the right polymer/solvent combination, prior to nanoparticle formation, that results in high protein drug loading is not straightforward. Polymer chains in solution fluctuate between extended or collapsed conformations and their average size can be measured using ensemble-averaged end-to-end distance or radius of gyration(R_g). Past literature studies have characterized polymer structure properties of PLGA-PEG in different solvents, but only a few have investigated whether or not varying polymer’s radius of gyration results in more favorable protein drug binding and influences the driving forces necessary for high protein drug loading. Therefore, this work uses atomistic molecular dynamics(MD) to examine the role of polymer extension on protein/polymer binding and its impact on the encapsulation process within PLGA-PEG nanoparticles.

Methods: In this work, protein/polymer interactions were evaluated by simulating 3 polymer oligomers at constant contour length, in the presence of a therapeutically relevant protein, Iduronate-2-sulphatase(ID2S), and pure water. This MD model was based on a simple back of the envelope calculation, using the experimental polymer and protein concentrations employed during nanoparticle formulation. 3 different initial system configurations were developed, in addition to testing three polymers (PLGA-PEG, PLGA and PEG) and three values of oligomer R_g. Hence, 27 total simulations were conducted in the NPT ensemble, for total production run time of 200 nanoseconds(ns). A harmonic potential was used to restrain all 3 oligomers’ R_g at high, medium, and low levels of extension for the first 100 ns to assess the effect of varying solvent quality on protein/polymer binding. After 100 ns, the harmonic restraint is turned off and the system is allowed to propagate for another 100 ns to gain some insights on whether binding is irreversible or PLGA prefers to collapse on itself in water. These systems were simulated in GROMACS using the AMBER99SB*-ILDNP forcefield to model the protein’s topological parameters, general amber forcefield(GAFF) for the polymers, and a three-point (TIP3P) explicit solvent model for the water. Analysis included: calculation of protein surface contact fraction as a measure of the degree of polymer interactions with the protein surface, frame occupancy as a measure of polymer residence time, surface patch/interaction interface ranking as a measure of interface hydrophobicity.

Results and Conclusions: Results showed that while the harmonic restraint was on, protein/PEG contacts were large at high and medium level of extension but small at low levels of extension. Surface patch ranking showed the polymer/protein interface was mostly polar and hydrophilic, when compared to other surface patches on ID2S. At the highest level of extension for the PLGA/protein system, high occupancy values and low contact fractions are found for ID2S surface residues, when compared to the other medium or low levels of extension. Moreover, the interface area was mostly hydrophobic, overlapping with patches containing few polar and charged residues. For the PLGA-PEG/protein system, there was a higher preference for the PEG block to be exposed to the solvent and for the PLGA block to be bound to ID2S surface. After the harmonic restraint was removed, polymer contact fraction increased for the medium and low levels of extension for the PEG/ID2S system and full collapse of the PLGA block was observed for low levels of extension, in the PLGA and PLGA-PEG/protein systems. Overall, insights from this study can help explain the localization of ID2S within PLGA-PEG nanoparticles, reducing the number of formulations needed to optimize protein loading.