Mapping Optimal Charge Density and Length of ROMP-Based PTDMs for siRNA Internalization

Abstract

A fundamental understanding of how polymer structure impacts internalization and delivery abilities of biologically relevant cargoes, particularly siRNA, is of critical importance to the successful design of improved delivery reagents. Herein we report the use of ring-opening metathesis polymerization (ROMP) methods to synthesize two series of guanidinium-rich protein transduction domain mimics (PTDMs): one based on an imide scaffold that contains one guanidinium moiety per repeat unit and another based on a diester scaffold that contains two guanidinium moieties per repeat unit. By varying both the degree of polymerization and, in effect, the relative number of cationic charges in each PTDM, the performances of the two ROMP backbones for siRNA internalization were evaluated and compared. Internalization of fluorescently labeled siRNA into Jurkat T cells demonstrated that FITC-siRNA internalization had a charge content dependence, with PTDMs containing approximately 40 to 60 cationic charges facilitating the most internalization. Despite this charge content dependence, the imide scaffold yielded much lower viabilities in Jurkat T cells than the corresponding diester PTDMs with similar numbers of cationic charges, suggesting that the diester scaffold is preferred for siRNA internalization and delivery applications. These developments not only improve our understanding of the structural factors necessary for optimal siRNA internalization, but will also guide the future development of optimized PTDMs for siRNA internalization and delivery.

Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters

“…Langer and coworkers have used high-throughput methods to find optimal lipid compositions for both cationic polymers (44)(45)(46) and LNPs (16,47,48). Additionally, Tew has shown that the lipid and charge distribution greatly influences siRNA uptake, especially in T lymphocytes (26,49). Taken together, these studies suggest that the uptake of the newly described CART-mRNA nanoparticles could be influenced by their lipid content, prompting our interest in a rapid method to identify preferred systems.…”

ROMP‐ and RAFT‐Based Guanidinium‐Containing Polymers as Scaffolds for Protein Mimic Synthesis

“…[41] In a similar system, it was found that an important interplay exists between cationic block length and delivery efficiency of siRNA into Jurkat T cells, with 20 repeat units of diguanidinium monomer being optimal. [40,47] In all of these cases, delivery was accomplished without covalent conjugation of cargo to the CPPM; a marked contrast to the CPPs, like TAT 49–57 . These structure-property studies are strong examples of progress that is enabled by new synthetic platforms and approaches.…”

“…Recently, a systematic study was conducted to determine the optimum hydrophobic window for the delivery of siRNA into Jurkat T cells . In a similar system, it was found that an important interplay exists between cationic block length and delivery efficiency of siRNA into Jurkat T cells, with 20 repeat units of diguanidinium monomer being optimal . In all of these cases, delivery was accomplished without covalent conjugation of cargo to the CPPM; a marked contrast to the CPPs, like TAT 49–57.…”

Increased block copolymer length improves intracellular availability of protein cargo

“…In much the same way that N : P ratio is held constant when optimizing the length of polymeric carriers for nucleic acid delivery, the ratio of polymer repeat units to protein molecules was maintained at 400 : 1 for all carriers. 35,37 As a result, the number of polymer chains in the system was decreased with increasing polymer length, effectively isolating the effect of monomer connectivity on carrier performance.…”

“…30–34 The effects of DP and charge density on siRNA delivery have been thoroughly explored using both cationic homopolymers and cationic-hydrophobic block copolymers, however, such optimization has thus far not been extended to protein delivery. 35–37 To date, ring-opening metathesis polymerization-based PTDMs have been used at a maximum length of just 20 repeat units with protein cargoes. 38,39…”