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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 of biologically relevant cargoes, particularly small interfering ribonucleic acid (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 fluorescein isothiocyanate (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 will 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.

SUBMITTER: Caffrey LM

PROVIDER: S-EPMC5094354 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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

Caffrey Leah M LM deRonde Brittany M BM Minter Lisa M LM Tew Gregory N GN

Biomacromolecules 20160921 10

A fundamental understanding of how polymer structure impacts internalization and delivery of biologically relevant cargoes, particularly small interfering ribonucleic acid (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 moiet ...[more]

PMID: 27599388

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Project description:Exploring the role of polymer structure for the internalization of biologically relevant cargo, specifically siRNA, is of critical importance to the development of improved delivery reagents. Herein, we report guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold containing tunable hydrophobic moieties that promote siRNA internalization. Structure-activity relationships using Jurkat T cells and HeLa cells were explored to determine how the length of the hydrophobic block and the hydrophobic side chain compositions of these PTDMs impacted siRNA internalization. To explore the hydrophobic block length, two different series of diblock copolymers were synthesized: one series with symmetric block lengths and one with asymmetric block lengths. At similar cationic block lengths, asymmetric and symmetric PTDMs promoted siRNA internalization in the same percentages of the cell population regardless of the hydrophobic block length; however, with 20 repeat units of cationic charge, the asymmetric block length had greater siRNA internalization, highlighting the nontrivial relationships between hydrophobicity and overall cationic charge. To further probe how the hydrophobic side chains impacted siRNA internalization, an additional series of asymmetric PTDMs was synthesized that featured a fixed hydrophobic block length of five repeat units that contained either dimethyl (dMe), methyl phenyl (MePh), or diphenyl (dPh) side chains and varied cationic block lengths. This series was further expanded to incorporate hydrophobic blocks consisting of diethyl (dEt), diisobutyl (diBu), and dicyclohexyl (dCy) based repeat units to better define the hydrophobic window for which our PTDMs had optimal activity. High-performance liquid chromatography retention times quantified the relative hydrophobicities of the noncationic building blocks. PTDMs containing the MePh, diBu, and dPh hydrophobic blocks were shown to have superior siRNA internalization capabilities compared to their more and less hydrophobic counterparts, demonstrating a critical window of relative hydrophobicity for optimal internalization. This better understanding of how hydrophobicity impacts PTDM-induced internalization efficiencies will help guide the development of future delivery reagents.

Project description:BACKGROUND:Spatial resolution in cardiac activation maps based on voltage measurement is limited by far-field interference. Precise characterization of electrical sources would resolve this limitation; however, practical charge-based cardiac mapping has not been achieved. METHODS:A prototype algorithm, developed from first principles of electrostatic field theory, derives charge density (CD) as a spatial representation of the true sources of the cardiac field. The algorithm processes multiple, simultaneous, noncontact voltage measurements within the cardiac chamber to inversely derive the global distribution of CD sources across the endocardial surface. RESULTS:Comparison of CD to an established computer-simulated model of atrial conduction demonstrated feasibility in terms of spatial, temporal, and morphologic metrics. Inverse reconstruction matched simulation with median spatial errors of 1.73 mm and 2.41 mm for CD and voltage, respectively. Median temporal error was less than 0.96 ms and morphologic correlation was greater than 0.90 for both CD and voltage. Activation patterns observed in human atrial flutter reproduced those established through contact maps, with a 4-fold improvement in resolution noted for CD over voltage. Global activation maps (charge density-based) are reported in atrial fibrillation with confirmed reduction of far-field interference. Arrhythmia cycle-length slowing and termination achieved through ablation of critical points demonstrated in the maps indicates both mechanistic and pathophysiological relevance. CONCLUSION:Global maps of cardiac activation based on CD enable classification of conduction patterns and localized nonpulmonary vein therapeutic targets in atrial fibrillation. The measurement capabilities of the approach have roles spanning deep phenotyping to therapeutic application. TRIAL REGISTRATION:ClinicalTrials.gov NCT01875614. FUNDING:The National Institute for Health Research (NIHR) Translational Research Program at Royal Papworth Hospital and Acutus Medical.

Dataset Information

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

Publications

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

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