Assistant Professor Villanova University, United States
Introduction:: Polymeric nanoparticles (NPs) are indispensable in the safe, sustained and controllable delivery of drugs. We have formulated spherical poly-amine-co-ester (PACE) polymeric NPs which can effectively deliver therapeutic nucleic acids (siRNA, mRNA) [1] to endothelial cells, a critical cell in the propagation of unwanted inflammation. The family of PACE polymers are copolymerized from a ratio 3 monomers which can be altered to affect physical changes to the resulting polymer, in particular the cation density [2]. Furthermore, the PACE NPs can be modified with different side chains and end groups. All of these changes affect drug loading and cell uptake. In this study, we are investigating changes to the polymer structure and end groups which increase amine concentration. We hypothesize this change will result in increased nucleic acid loading, cell uptake, and endosomal escape, due to increased ionizable groups and electrostatic interactions.
Materials and Methods:: A family of PACE polymers were synthesized from various ratios of 15-pentadecanolide (PDL) and N-methyldiethanolamine (MDEA) (PDL:MDEA = 50%:50%; 60%:40%; 70%:30%) and modified with end groups (carboxylic acid (acid), polyethylene glycol (PEG), N-(2-Aminoethyl)-1,3-propanediamine (NAP). PACE polymer NPs encapsulating fluorescent dyes and short-chain nucleic acid oligomers are formed through a double emulsion. In some cases, a surfactant was used to stabilize the emulsion (poly-vinyl alcohol). Dynamic light scattering (DLS) and zeta potential were used to characterize the average size and surface charge of PACE NPs. Scanning electron microscope (SEM) and atomic forced microscopy (AFM) were used for characterizing the morphology, and electrostatic force microscopy (EFM) were used for mapping the positive charge on the surface. The loading efficiency of cargo was determined with a picogreen assay, and TNS assay was conducted for determination of dsDNA encapsulation and pKa values on the PACE NPs. Human umbilical vein endothelial cells (HUVECs) are cultured and incubated under 5% CO2 and 37C and flow cytometry is used for quantifying the cell uptake rate of different PACE NPs based on the characterized surface changes.
Results, Conclusions, and Discussions:: PACE NPs can be formulated from polymers made with various ratios of monomers and this change affects the resulting NP’s surface charge. We have shown that within a set monomer ratio, the polymers can be further modified with end groups which affect surface charge of the polymer, nucleic acid encapsulation efficiency, and cell uptake rates and cytotoxicity. Shown in Figure 1 is a subset of PACE60 NPs with various modifications, demonstrating the effect on surface changes (charge and pKa). The morphology of selected NPs, displayed by SEM (Fig.1A), are spherical with average diameter size of 180-300nm, with changes corresponding to size of end group modification (i.e., NPs with PEG are largest). Charges are distributed over the surface of NPs (mapped by EFM, Fig. 1B) due to positive charged polymer loaded dsDNA organization. Fig.1C shows the pKa values of different types of PACENPs. These values range from 5.1 to 5.8 and are primarily governed by the monomer ratio of the polymer (i.e., PACE50 vs PACE70). However, the pKa can be altered with the addition of amines through end group modification as shown. The changes to polymer charge density also effect nucleic acid loading, with increased loading corresponding to additional amine groups incorporated.
The PACE NPs with various ratios of monomers can affect the surface charge values. The polymer can also be modified further by adding different end groups to affect the surface charge. The change of surface charge values and distribution of surface charge for PACE NPs will further influence the efficiency of nucleic acid encapsulation and endothelial cell uptake rates and cytotoxicity, and can be tuned to optimized cell uptake with minimal toxicity.
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References (Optional): : 1. Yin, J., Piotrowski-Daspit, A. S., Zhang, J., Shao, M., Bracaglia, L. G., Utsumi, T., Seo, Y., DiRito, J., Song, E., Wu, C. Q., Inada, A., Tietjen, G. T., Pober, J. S., Iwakiri, Y., & Saltzman, W. M. (2019b). Poly(amine-co-ester) nanoparticles for effective Nogo-B knockdown in the liver. Journal of Controlled Release, 304, 259–267. https://doi.org/10.1016/j.jconrel.2019.04.044
2. Kauffman, A. B., Piotrowski-Daspit, A. S., Nakazawa, K. H., Jiang, Y., Datye, A., & Saltzman, W. M. (2018). Tunability of Biodegradable Poly(amine-co-ester) Polymers for Customized Nucleic Acid Delivery and Other Biomedical Applications. Biomacromolecules, 19(9), 3861–3873. https://doi.org/10.1021/acs.biomac.8b00997
