Trehalose Click Polymers Inhibit Nanoparticle Aggregation and Promote pDNA Delivery in Serum

Abstract

Herein, three new glycopolymers have been synthesized via "click polymerization" to promote nucleic acid delivery in the presence of biological media containing serum. These structures were designed to contain a trehalose moiety to promote biocompatibility, water solubility, and stability against aggregation, amide-triazole groups to enhance DNA binding affinity, and an oligoamine unit to facilitate DNA encapsulation, phosphate neutralization, and interactions with cell surfaces. A 2,3,4,2',3',4'-hexa-O-acetyl-6,6'-diazido-6,6'-dideoxy-D-trehalose (4) monomer was polymerized via copper(I)-catalyzed azide-alkyne cycloaddition with a series of dialkyne-amide comonomers that contain either one, two, or three Boc-protected secondary amines (7a, 7b, or 7c, respectively). After deprotection, three water-soluble polycations (9a, 9b, or 9c) were obtained with similar degrees of polymerization (n = 56-61) to elucidate the role of amine number on nucleic acid binding, complex formation, stability, and cellular delivery. Gel electrophoresis and ethidium bromide experiments showed that 9a-9c associated with plasmid DNA (pDNA) and formed complexes (polyplexes) at N/P ratios dependent on the amine number. TEM experiments revealed that 9a-9c polyplexes were small (50-120 nm) and had morphologies (spherical and rodlike) associated with the polymer chain stiffness. Dynamic light scattering studies in the presence of media containing serum demonstrated that 9c polyplexes had a low degree of flocculation, whereas 9a and 9b polyplexesd aggregate rapidly. Further biological studies revealed that these structures were biocompatible and deliver pDNA into HeLa cells. Particularly, 9c polyplexes promoted high delivery efficacy and gene expression profiles in the presence of serum.

Polymer beacons for luminescence and magnetic resonance imaging of DNA delivery

“…Polymers Eu3a, Eu3b, Gd3a, and Gd3b began to charge-neutralize pDNA at an N/P ratio of 5, and pDNA migration was mostly suppressed at N/P of 20. With these polymers, the N/P ratio needed to fully inhibit gel migration of pDNA was higher than other polyamidoamine vehicles created in our laboratory (5). The presence of the carboxylate groups likely lowers the cationic charge on the polymer backbone.…”

“…All other reagents and solvents used in the synthesis were obtained from Aldrich and were used without further purification. Monomers 1a and 1b were synthesized following a previously published procedure developed in our lab (5). DTPA-bisanhydride (BA) was prepared using a standard procedure.…”

“…A solution of 0.5 M sodium acetate containing 20% acetonitrile was used as the mobile phase. Agarose gel electrophoreses shift assays and TEM studies (Philips EM 420 Scanning TEM) were performed according to previously published methods (4,5).…”

“…Cells were aspirated of old media, washed with 1 mL PBS, and the appropriate polyplex solution was added to each well. Cells were then incubated at 37°C under a 5% CO 2 atmosphere for 4 h. The cell suspensions were then prepared for analysis as previously described (5). HeLa cell suspensions were analyzed on a FACS Canto II flow cytometer equipped with a 633 nm helium-neon laser.…”

“…The use of synthetic materials, such as polymers, for polynucleotide delivery has rapidly grown, and presents a wealth of promising alternatives to conventional viral vectors (3)(4)(5), which have caused serious problems in the clinic. Although much is known about the infection pathways, advantages, and troubles of viral vectors (6), researchers in the field of nonviral delivery are just beginning to understand the transfection mechanisms, benefits, and potential issues with the multitude of materials being developed as macromolecular drug carriers (7,8).…”