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Relating structure and internalization for ROMP-based protein mimics.

ABSTRACT: 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. 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. Specifically, several guanidine rich homopolymers, along with an amphiphilic block copolymer were used to investigate the relationship between structure and internalization activity in HeLa cells, both alone and non-covalently complexed with EGFP by flow cytometery and confocal imaging. The findings indicate that while changing the amount of positive charge on our PTDMs does not seem to affect the endosomal uptake, the presence of hydrophobicity appears to be a critical factor for the polymers to enter cells either alone, or with associated cargo.

SUBMITTER: Backlund CM

PROVIDER: S-EPMC4964965 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Relating structure and internalization for ROMP-based protein mimics.

Backlund Coralie M CM Takeuchi Toshihide T Futaki Shiroh S Tew Gregory N GN

Biochimica et biophysica acta 20160331 7 Pt A

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. 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. Specifically, sev ...[more]

PMID: 27039278

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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.

Dataset Information

Relating structure and internalization for ROMP-based protein mimics.

Publications

Relating structure and internalization for ROMP-based protein mimics.

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