(M1530-08-48) End Group Chemistry Modulates Physical Properties and Biomolecule Release from Biodegradable Polyesters

Purpose: Long-acting injectable protein therapeutics are a rapidly advancing arm of pharmaceuticals. A promising and versatile class of such formulations involves encapsulation of therapeutic protein within poly(lactic-co-glycolic acid) (PLGA) degradable microparticles (MP) to shield the protein from enzymatic degradation and control the release rate¹. However, models based on degradation and erosion of PLGA polymer matrices do not always fully capture release behavior, due in part to electrostatic interactions between the polymer terminal group and encapsulated compound². The repertoire of functionalized PLGA polymers commercially available has now expanded to include terminal group chemistries that may significantly alter polymer characteristics including charge, hydrophobicity, and erosion^{3, 4}. This work aims to explore how PLGA terminal group chemistry affects polymer physical properties and charged biomolecule release kinetics. PLGA with hydroxyl (PLGA-OH), amine (PLGA-NH₂), or carboxylic acid (PLGA-COOH) terminal groups that have neutral, positive, or negative charge, respectively, were evaluated.

Methods: The physical properties of PLGA polymers were assessed including hydrophobicity, viscosity, and glass transition temperature. Microparticles (MP) were fabricated from these polymers and their morphology and degradation rates were quantified via scanning electron microscopy (SEM) and size exclusion chromatography (SEC), respectively. Additionally, the pH change associated with PLGA MP degradation was quantified. MP were co-cultured with bone marrow derived macrophages to assess potential to alter macrophage phenotype as quantified by flow cytometry and internalization as quantified by ImageStream cytometry. Release rates of encapsulated model peptides and proteins were determined via spectrophotometry and enzyme-linked immunosorbent assay.

Results: Experiments assessing the physical properties of the polymers indicate PLGA-NH₂ has reduced hydrophobicity, degrades faster, exhibits emulsion stabilizing behavior, and has reduced phagocytic clearance by bone marrow derived macrophages. Charged biomolecule release rates are increased from PLGA-NH₂ MPs and slightly accelerated from PLGA-OH MPs, compared to PLGA-COOH MPs.

Conclusion: These studies provide further insight into the interactions between charged biomolecules and the encapsulating polymer and could provide additional tools to tune release for various protein therapeutics that experience such interactions. Importantly, this work demonstrates that small chemical changes to polymer structures, namely the end-cap functional group, has the potential to impart significant changes to polymer physical properties and the release rate of biomolecules from long acting injectable microparticles.

References: 1. Werle, M.; Bernkop-Schnurch, A., Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids 2006, 30 (4), 351-67.
2. Blanco, M. D.; Alonso, M. J., Development and characterization of protein-loaded poly(lactide-co-glycolide) nanospheres. European Journal of Pharmaceutics and Biopharmaceutics 1997, 43 (3), 287-294.
3. Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad, O. C., Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery. Biomaterials 2007, 28 (5), 869-76.
4. El-Hammadi, M. M.; Arias, J. L., Recent Advances in the Surface Functionalization of PLGA-Based Nanomedicines. Nanomaterials (Basel) 2022, 12 (3).

Acknowledgements: We wish to acknowledge the helpful sharing of resources and data interpretation that Dr. Tara Myers’ research team at the University of Pittsburgh provided for the analysis of PLGA molecular weight via SEC Work performed in the University of Pittsburgh Nanofabrication and Characterization Core Facility (RRID:SCR_05124) and services and instruments used in this project were graciously supported, in part, by the University of Pittsburgh.

Amine functionalized PLGA displays altered physical properties

PLGA polymers terminated with carboxylic acid (-COOH), amine (-NH2), and hydroxyl (-OH) groups were characterized. (A) Representative images and (B) quantitative analysis of water droplet contact angle on polymer films shows PLGA-NH2 reduces the surface tension (contact angle) of water (N = 30). (C) Representative DSC analysis for -20 to 150 °C at 10°C/min ramp for each of the PLGA polymers is shown, in which the circle denotes the glass transition temperature (Tg). (D) The measured Tg from N = 3 independent experiments shows reduced Tg for PLGA-NH2. Statistical comparisons were made using one-way ANOVA followed by Šídák's multiple comparisons test *** p ≤ 0.001, **** p ≤ 0.0001.


Representative images of microparticles internalized by BMDMΦs

Bright field microscope 1 (BF1) shows cell morphology and internalized microparticles. Fluorescent intentsity is shown for internalized microparticles (MP) loaded with Alexa Fluor 680 labeled dextran (681/704; ex/em), the nucleus (DAPI), and the cell membrane via CD11b staining (FITC). An colored overlay image is shown with the following weight to each image: BF1 – 20%, Nucleus – 80%, CD11b – 100%, MP – 100%.

Biomolecule release & encapsulation with end-cap PLGA shows charge-dependent behavior

Cumulative release of positively and neutrally charged peptide from porous MPs with altered PLGA end caps was quantified. (A) size distributions of microparticles loaded with the neutral peptide, positive peptide, and CCL22 protein. Supplementary Table XX details the mean and standard deviation of the size distribution for each formulation. (B) Neutral peptide release kinetics demonstrate biomolecule release in the absence of charge interactions. (C) Positively charged peptide release is also influenced by peptide-polymer electrostatic interactions. (D) For the larger and more complex protein, CCL22, the order of release is consistent with that for the positive peptide, but release from PLGA-OH and PLGA-COOH MPs is significantly depressed compared to PLGA-NH2 MPs. Data in A represent volume impedance measurement of particle size for N = 10,000 events. Data in B-D represent mean + sd for N = 3 independent release experiments.