Design of Aromatic‐Containing Cell‐Penetrating Peptide Mimics with Structurally Modified π Electronics

deRonde, Brittany M.; Birke, Alexander; Tew, Gregory N.

doi:10.1002/chem.201405381

Cited by 34 publications

(42 citation statements)

References 61 publications

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“…Using R8 as inspiration and a benchmark, a 28,31–36 set of four homopolymers and one block copolymer (Figure 1) were chosen to investigate the impact of polymer structure and backbone on internalization efficiency in HeLa cells.…”

Section: Introductionmentioning

confidence: 99%

Relating structure and internalization for ROMP-based protein mimics

Backlund

Takeuchi

Futaki

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Elucidating the predominant cellular entry mechanism for protein transduction domains (PTDs) and their synthetic mimics (PTDMs) is a complicated problem that continues to be a significant source of debate in the literature. Several guanidinium-rich homopolymer structures initially designed to mimic oligoarginine, as well as an amphiphilic block copolymer, were end-labeled with FITC. This enabled the monitoring of PTDM internalization into HeLa cells by flow cytometry and confocal imaging. Additionally, their unlabeled counterparts showed improved ability to deliver proteins into cells with added hydrophobic content. In conjunction, pre-incubation with the protein is required, suggesting that the polymers are not just simply interacting with the membrane, but require association with the cargo of interest. However, the mechanism of cellular entry is not dependent on structure within this study, as punctate fluorescence was prevalent within the cells treated with fluorescently labeled samples and protein-polymer complexes. This suggests that the predominant mode of internalization for the presented PTDM structures is endosomal uptake and does not appear to be affected by concentration or structure. The PTDMs reported here provide a well-controlled platform to vary molecular composition for structure activity relationship studies to further our understanding of PTDs, their non-covalent association with cargo, and their cellular internalization pathways.

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Section: Introductionmentioning

confidence: 99%

Relating structure and internalization for ROMP-based protein mimics

Backlund

Takeuchi

Futaki

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…To this end, our research group has devoted an extensive amount of research into understanding how the structures of ring-opening metathesis (ROMP) based protein mimics influence their membrane interactions 29,40,44–48 , cellular uptake efficiencies 29,49 , and siRNA delivery. 30,32 We have demonstrated the utility of the platform for the successful internalization of siRNA and for the knockdown of active biological genes in T cells.…”

Section: Introductionmentioning

confidence: 99%

Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization

et al. 2016

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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 twenty repeat units of cationic charge, the asymmetric block length had greater siRNA internalization, highlighting the non-trivial 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. HPLC retention times quantified the relative hydrophobicities of the non-cationic 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.

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“…19,49,50 Initial studies aimed to understand the effects of PTDM length 51 , hydrophobicity 52 , aromaticity 53 , aromatic π-electronics 54 , and sequence segregation of cationic/hydrophobic components 55 on membrane interactions and cellular uptake. 43 In early 2013, we extended this platform to include siRNA delivery.…”

Section: Introductionmentioning

confidence: 99%

Development of Guanidinium-Rich Protein Mimics for Efficient siRNA Delivery into Human T Cells

et al. 2015

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RNA interference is gaining attention as a means to explore new molecular pathways and for its potential as a therapeutic; however, its application in immortal and primary T cells is limited due to challenges with efficient delivery in these cells types. Herein, we report the development of guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold that delivers siRNA into Jurkat T cells and human peripheral blood mononuclear cells (hPBMCs). Homopolymer and block copolymer PTDMs with varying numbers of guanidinium moieties were designed and tested to assess the affect cationic charge content and the addition of a segregated, hydrophobic block had on siRNA delivery. Delivery of fluorescently-labeled siRNA into Jurkat T cells illustrates that the optimal cationic charge content, 40 charges per polymer, leads to higher efficiencies, with block copolymers outperforming their homopolymer counterparts. PTDMs also outperformed commercial reagents commonly used for siRNA delivery applications. Select PTDM candidates were further screened to assess the role PTDM structure has on the delivery of biologically active siRNA into primary cells. Specifically, siRNA to hNOTCH1 was delivered to hPBMCs enabling 50–80% knockdown efficiencies, with longer PTDMs showing improved protein reduction. By evaluating the PTDM design parameters for siRNA delivery, more efficient PTDMs were discovered that improved delivery and gene knockdown in T cells. Given the robust delivery of siRNA by these novel PTDMs, their development should aid in the exploration of T cell molecular pathways leading eventually to new therapeutics.

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Design of Aromatic‐Containing Cell‐Penetrating Peptide Mimics with Structurally Modified π Electronics

Cited by 34 publications

References 61 publications

Relating structure and internalization for ROMP-based protein mimics

Relating structure and internalization for ROMP-based protein mimics

Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization

Development of Guanidinium-Rich Protein Mimics for Efficient siRNA Delivery into Human T Cells

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