Development of Guanidinium-Rich Protein Mimics for Efficient siRNA

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Article

Development of Guanidinium-rich Protein Mimics for Efficient siRNA Delivery into Human T Cells Brittany Morgan deRonde, Joe A. Torres, Lisa M Minter, and Gregory N. Tew Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00795 • Publication Date (Web): 31 Aug 2015 Downloaded from http://pubs.acs.org on September 11, 2015

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Development of Guanidinium-rich Protein Mimics

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for Efficient siRNA Delivery into Human T Cells

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Brittany M. deRonde†, Joe A. Torres§,‡, Lisa M. Minter§,‡, and Gregory N. Tew*,†,§,‡

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†

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Amherst, MA 01003

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§

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Amherst, MA 01003

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‡

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01003

Department of Polymer Science and Engineering, University of Massachusetts Amherst,

Department of Veterinary and Animal Sciences, University of Massachusetts Amherst,

Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst, MA

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ABSTRACT. RNA interference is gaining attention as a means to explore new molecular

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pathways and for its potential as a therapeutic; however, its application in immortal and primary

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T cells is limited due to challenges with efficient delivery in these cells types. Herein, we report

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the development of guanidinium-rich protein transduction domain mimics (PTDMs) based on a

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ring-opening metathesis polymerization scaffold that delivers siRNA into Jurkat T cells and

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human peripheral blood mononuclear cells (hPBMCs). Homopolymer and block copolymer

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PTDMs with varying numbers of guanidinium moieties were designed and tested to assess the

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affect cationic charge content and the addition of a segregated, hydrophobic block had on siRNA

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delivery. Delivery of fluorescently-labeled siRNA into Jurkat T cells illustrates that the optimal

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cationic charge content, 40 charges per polymer, leads to higher efficiencies, with block

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copolymers outperforming their homopolymer counterparts.

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commercial reagents commonly used for siRNA delivery applications. Select PTDM candidates

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were further screened to assess the role PTDM structure has on the delivery of biologically

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active siRNA into primary cells. Specifically, siRNA to hNOTCH1 was delivered to hPBMCs

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enabling 50-80% knockdown efficiencies, with longer PTDMs showing improved protein

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reduction. By evaluating the PTDM design parameters for siRNA delivery, more efficient

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PTDMs were discovered that improved delivery and gene knockdown in T cells. Given the

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robust delivery of siRNA by these novel PTDMs, their development should aid in the

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exploration of T cell molecular pathways leading eventually to new therapeutics.

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INTRODUCTION:

PTDMs also outperformed

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RNA interference (RNAi), discovered almost two decades ago, continues to be an

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important tool to probe molecular pathways and to potentially treat diseases.1-5 Small interfering

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RNA (siRNA), which represents one RNAi approach, must be present in the cytosol so that the

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siRNA guide strand can be incorporated into the RISC complex and degrade its complementary

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mRNA.

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which is advantageous for discrete biological and clinical applications,1-5 a strategy of critical

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importance in the context of the immune system and T cells.9-12

This leads to transient, sequence-specific, post-transcriptional gene knockdown,6-8

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T cells orchestrate essential functions during the immune response to pathogens, chronic

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inflammation, and autoimmune disorders.10 Harnessing the capabilities of RNAi would allow

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immunologists to explore molecular pathways leading to a better understanding of T cell


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activation, signaling, and other biological processes, eventually providing new treatment options

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for autoimmune diseases.9-12 Unfortunately, progress in this area has been severely limited due

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to the lack of robust delivery technologies for T cell lines and primary cells.10,12-17 Three major

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strategies are routinely used for siRNA delivery into T cells and primary cells: electroporation,

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viral vectors, and transfection.12-17 The use of electroporation and viral vectors both have severe

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drawbacks, which include high cell death and potential mutagenic/immunogenic effects,

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respectively.14,16 Transfection is generally a safer alternative, with higher cell survival rates;

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however, many current delivery vehicles exhibit low efficiencies in T cell lines and primary

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cells.13,15,17 Although designing successful carriers for T cells has been a challenge, highly

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modular protein transduction domain mimics (PTDMs), sometimes referred to as cell-penetrating

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peptide mimics (CPPMs), have been used successfully in other more easily transfected cell types

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and can provide insight for the design of more efficient delivery vehicles.18-20

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Inspiration for PTDMs is derived from proteins with translocation abilities, such as the

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HIV-1 TAT and Antennapedia homeodomain proteins, as well as other protein transduction

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domains (PTDs) and cell-penetrating peptides (CPPs) that exhibit delivery capabilities.21-23

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PTDMs incorporate important features of PTDs and CPPs critical for intracellular delivery24-29,

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including cationic charge content provided by guanidinium groups, and partial hydrophobicity

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contributed by the backbone architecture and/or the incorporation of hydrophobic monomers.18,19

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Although some PTDs and CPPs, such as CADY and MPG, possess these key features and have

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already been designed and commercialized for siRNA delivery, PTDMs offer many distinct

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advantages over their peptide counterparts.30,31 Moving away from a peptide-based architecture

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provides protection from proteolysis and avoids solid phase peptide synthesis. Additionally, a

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non-peptidic system offers many more structural options, since it is not restricted to the


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incorporation of known amino acids.18,19,32 Consequently, different chemistries can be used to

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design molecules, and chemical compositions can be tuned more widely to improve delivery of

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specific cargo.18,19,32

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antimicrobial peptide mimics, where the key features, including their facially amphiphilic

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architecture,33-35 were incorporated into synthetic scaffolds to yield new antimicrobial

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agents.19,35-37

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This design rationale has proven successful in creating more potent

In the realm of delivery reagents, several groups have demonstrated the utility of

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synthetic, guanidinium-rich, polymeric scaffolds to deliver siRNA.18,19,38-44

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metathesis

(ROMP)20,45-47,

polymerization

polymethacrylamide42,48,

Ring-opening arginine-grafted

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bioreducible polydisulfide40,41, and oligocarbonate38,39,44 scaffolds have all been developed and

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screened for siRNA delivery. The successful design of PTDMs will further the understanding of

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the key features necessary for efficient delivery, as well as enable the development of more

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effective delivery reagents.18,19,32

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Related to this long term goal, in 2008, the Tew research group first reported the

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development of polymeric guanidinium-rich PTDMs based on a ROMP scaffold that mimics

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PTDs/CPPs, such as TAT49-57 and oligoarginine (R9).19,49,50 Initial studies aimed to understand

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the effects of PTDM length51, hydrophobicity52, aromaticity53, aromatic π-electronics54, and

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sequence segregation of cationic/hydrophobic components55 on membrane interactions and

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cellular uptake.43 In early 2013, we extended this platform to include siRNA delivery. It was

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demonstrated that our PTDMs delivered FITC-siRNA into Jurkat T cells with efficiencies greater

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than 90% and achieved 50% knockdown of NOTCH1 protein in human T cells from human

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peripheral blood mononuclear cells (hPBMCs).20 Due to the initial success of siRNA delivery


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using these PTDMs, further understanding of the relationship between PTDM structure and

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siRNA delivery efficiency in T cells was desired.

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Herein we document our efforts to tune ROMP-based PTDM structures for improved

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siRNA delivery. Structure activity relationships using Jurkat T cells and hPBMCs were used to

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probe how polymer charge content and the addition of a segregated, hydrophobic block impacted

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siRNA internalization and delivery efficiencies.

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developed, homopolymers and block copolymers, with matching cationic charge contents

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(Figure 1). All block copolymers contained a constant hydrophobic block of five repeat units.

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It should be noted that guanidinium moieties were incorporated to mimic arginine residues and

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phenyl moieties were incorporated in the block copolymer PTDMs to mimic phenylalanine

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residues, both of which have been shown to play critical roles in membrane interactions.27,29,43,51-

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54,57,58

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PTDMs, which can be used to develop the next generation of highly efficient transfection

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reagents.

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EXPERIMENTAL SECTION

Specifically, two polymer series were

In this study, we elucidate the essential design parameters for siRNA delivery using our

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Monomer Synthesis and Characterization. Monomers were synthesized using a two-step

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process adapted from Lienkamp et al. with modifications.59 In brief, oxanorbornene anhydride

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was ring-opened using the desired alcohol and 4-dimethylaminopyridine (DMAP) as a catalyst to

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yield the half-ester intermediates, which precipitated from solution. The half-ester was then

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further

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dimethylaminopropyl)carbodiimide (EDC) coupling to yield the monomer. A one-pot synthesis

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was used for monomers designed to display two of the same functionalities. All monomers were

reacted

with

the

second

desired

alcohol

using

1-ethyl-3-(3-


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purified using column chromatography (ethyl acetate/dichloromethane (CH2Cl2), 10/90, v/v) and

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subsequently analyzed by

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spectrometry (MS) to assess their chemical compositions and purity.

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procedures and all characterization data are provided in the supporting information (Section II,

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Figures S1 and S2).

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PTDM Synthesis and Characterization. Homopolymers and block copolymer PTDMs were

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synthesized by ROMP using Grubbs’ third generation catalyst following previously described

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methods.20,59 In brief, degassed monomer solutions in CH2Cl2 were introduced to degassed

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catalyst solutions in CH2Cl2 and allowed to stir.

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H NMR spectroscopy,

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C NMR spectroscopy, and mass Detailed synthetic

For block copolymers, the hydrophobic

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monomer was added first followed by the Boc-protected guanidinium monomer. To prevent

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premature termination of polymerizations, all guanidinium-based monomers were Boc-protected

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to limit the potential for catalyst coordination. The presence of the Boc groups also allowed for

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sufficient solubility in organic solvents.

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precipitated, and subsequently deprotected using a 1:1 ratio of CH2Cl2:trifluoroacetic acid

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(TFA). Excess TFA was removed by azeotropic distillation with methanol. Polymers were then

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dissolved in water and transferred to Biotech CE dialysis tubing membranes with a MWCO 100-

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500 g/mol and dialyzed against RO water until the conductivity of the water remained < 0.2 µS

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(2-3 days). All polymers were characterized by 1H NMR and gel permeation chromatography

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(GPC) to assess chemical compositions and molecular weight distributions, respectively.

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Detailed synthetic procedures and all characterization data are provided in the supporting

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information (Section II, Figures S3-S12).

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Cell culture (Cell lines) and FITC-siRNA Internalization. Jurkat T cells and HeLa cells were

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cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% (v/v) FBS, 100 U/mL

Polymers were quenched with ethyl vinyl ether,


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non-essential amino acids, 100U/mL sodium pyruvate, 100 U/mL penicillin, and 100 U/mL

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streptomycin. Jurkat T cells were incubated at 37 °C with 5% CO2 and passaged 24 hours prior

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to experimentation. On the day of the experiment, PTDMs were mixed with siRNA at an N:P

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ratio of 8:1 (50 nM siRNA/well) and allowed to incubate at room temperature for 30 minutes

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then added drop-wise to the cells (4x105 cells/well; 1 mL total volume) in a 12 well plate. Cells

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were incubated at 37 °C with 5% CO2 in serum-containing media for four hours prior to analysis

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by flow cytometry. For HeLa cell experiments, cells (5x104 cells/well; 1 mL total volume) in

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serum containing media were cultured in 12 well plates for 48 hours so that the cells would be

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70-90% confluent on the day of the experiment. On the day of the experiment, PTDMs were

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mixed with siRNA at an N:P ratio of 4:1 (50 nM siRNA/well) and allowed to incubate at room

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temperature for 30 minutes. The cell media was replaced with fresh, complete media prior to

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adding the PTDM/siRNA complexes carefully to the top of the sample wells. Cells were

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incubated at 37 °C with 5% CO2 for four hours in serum-containing media prior to analysis by

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flow cytometry. Cell viability was assessed using 7-AAD/Annexin V staining.

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Primary Cell Enrichment and Stimulation. hPBMCs, purchased from Stemcell Technologies,

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Inc. in 2.5x106 cells/aliquot (Product # 70047.2) and obtained by the company using institutional

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review board approved consent forms and protocols, were thawed and plated in a 6 well plate

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using RPMI 1640 supplemented with 10% (v/v) FBS, 100 U/mL non-essential amino acids, 100

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U/mL sodium pyruvate, 100 U/mL penicillin, and 100 U/mL streptomycin, to enrich for the T

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cell populations. The cells were incubated at 37 °C in a 5% CO2 atmosphere overnight. On the

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day of the experiment, PTDMs were mixed with siRNA at an N:P ratio of 8:1 (100 nM siRNA

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per well) and allowed to incubate at room temperature for 30 minutes prior to adding them drop-

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wise to the cells (1x106 cells/well) in a 24 well plate. Cells were incubated at 37 °C with 5%


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CO2 in serum-containing media for four hours and subsequently harvested, washed, re-plated in

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an anti-CD3 and anti-CD28 coated well plate (well plate coated the night before) to stimulate the

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cells. Cells were incubated at 37 °C with 5% CO2 in serum-containing media for 48 hours prior

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to harvesting for experiments. For flow cytometry, half the cells were fixed for intracellular

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staining of NOTCH1 protein and the other half were used for viability staining. Detailed cellular

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assay procedures as well as viability data and flow cytometry histograms are provided in the

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supporting information (Section V).

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Assessment of NOTCH1 Knockdown in hPBMCs. Following cell harvest and wash steps,

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cells were re-suspended in 100 µL of the Foxp3 Fix/Perm Cocktail and incubated for 30 min on

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ice protected from light. After the 30 min incubation, the cells were brought up to 200 µL with

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the permeabilization wash buffer Cells were washed three times with the permeablization wash

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buffer. After the third wash step, cells were re-suspended in 50 µL of the permeablization wash

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buffer, stained with 2 µL of anti-human NOTCH1 PE, and incubated for 30 min on ice protected

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light. After the 30 min incubation, cells were washed three times with the permeablization wash

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buffer and then re-suspended in 200 µL of FACS wash buffer and transferred to FCM tubes for

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analysis. For flow cytometry analysis, the fluorescence signal was collected for 10,000 cells.

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The cell populations were gated in order to assess the percent of positive cells, which reflected

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the percentage of the cell population expressing hNOTCH1 protein.

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represented the amount of hNOTCH1 protein present in the cells. The percent relative protein

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expression represents the percent positive cells multiplied by the MFI, normalized to the blank,

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and multiplied by 100%.

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Assessment of hPBMC Viability. Following the wash steps documented above, cells were re-

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suspended in 200 µL of a 7-AAD stock solution (2.4 mL of binding buffer + 60 µL of 7-AAD

The calculated MFI


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stain; Solution was scaled up or down as needed) and transferred to FCM tubes for analysis. For

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flow cytometry analysis, the fluorescence signal was collected for 10,000 cells. The cell

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populations were gated in order to assess the percent of positive cells, which reflected the

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percentage of dead cell in the population.

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RESULTS AND DISCUSSION

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PTDM Design and Characterization

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Proteins and peptides have been utilized extensively for intracellular delivery

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applications.18,32,60-62 These materials, however, have presented many limitations, including long

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and costly synthetic procedures, as well as poor stability, all of which can be avoided by

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leveraging more versatile synthetic platforms.

In this report, ROMP with Grubbs’ third

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generation catalyst was used to synthesize all PTDMs since it is a fast, efficient, and functional

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group tolerant method that also allows for good control over molecular weights and dispersities..

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The living nature of many ROMP polymerizations also enables the synthesis both of

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homopolymers and block copolymers, and allows the influence of an added hydrophobic block

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on internalization and delivery efficiencies to be evaluated within the same monomer

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chemistry.63-71

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quite versatile, allowing for the incorporation of the same or different functionalities to tune the

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polymer structures at the monomer level.18 For this particular endeavor, a monomer containing

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two Boc-protected guanidinium groups (dG; Figure 1) and a second monomer containing a

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methyl group and a phenyl group (MePh; Figure 1) were synthesized. Guanidinium moieties

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were selected as the cations because they have been previously shown to yield superior uptake

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and delivery efficiencies as compared to their ammonium counterparts found in lysine and

Additionally, the oxanorbornene-based dual-functional monomer platform is


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ornithine.27 The number average molecular weight (Mn) for the homopolymerization of the dG

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monomer increased linearly with monomer to initiator ratio ([M]/[I]), while maintaining low

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dispersities. This indicated that it polymerized in a controlled fashion and can be used to

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synthesize both homopolymers and block copolymers (Figure S12).

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Figure 1. Monomer and polymer structures used for this study. A) Monomer structures. B) Polymer structures. C) Table summarizing the polymer nomenclature and the corresponding number of positive charges each polymer contains. Blue represents cationic moieties and green represents hydrophobic moieties.

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Homopolymer and block copolymer PTDMs were synthesized to determine how the

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cationic charge content, as well as the incorporation of a segregated hydrophobic region,

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impacted siRNA delivery. To that end, PTDMs with increasing amounts of cationic charge were

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synthesized, as documented in Figure 1. Since the cationic monomer contains two guanidinium

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groups, the number of charges is reported as twice the degree of polymerization (Figure 1C). In


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addition, block copolymer PTDMs were designed with segregated hydrophobic and cationic

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domains, as shown in Figure 1. These were specifically designed because the incorporation of a

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hydrophobic domain has been shown to improve internalization and delivery efficiencies through

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interactions with hydrophobic lipids.31 The performance of the block copolymers was compared

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to their corresponding homopolymer derivatives, which contained the same charge content, in

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order to assess the effect of incorporating a hydrophobic block. In both cases, the charge content

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was varied up to 160 charges (80 repeat units); however, PTDMs with 160 charges had limited

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solubility and were therefore not used for any biological studies. Overall, this series of PTDMs

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provided insight into key design parameters for efficient siRNA internalization and delivery.

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FITC-siRNA Delivery

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Preliminary studies were conducted using fluorescein isothiocyanate (FITC) labeled

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siRNA (FITC-siRNA), to assess trends in internalization efficiencies in Jurkat T cells resulting

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from differences in PTDM charge content and the presence or absence of a hydrophobic block.

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The fluorescent label on the siRNA allowed cell populations to be analyzed using flow

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cytometry. Jurkat T cells were selected because they are typically difficult to transfect and

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manipulate.10,12-17 The N:P ratio used for complex formation between PTDMs and siRNA was

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8:1 and was established by screening FITC-siRNA internalization efficiencies as a function of

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N:P ratio in addition to using gel retardation assays to assess siRNA complexation (Figures S13-

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S22 and Figure S25). This N:P ratio used was consistent with our previous publication.20 Gel

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retardation assays for PTDM/siRNA complexes at N:P ratios of 0.5:1, 1:1, 2:1, 4:1, 8:1, and 12:1

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are included in the supporting information (Figures S13-S21). In brief, it was demonstrated that

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shorter polymers (40 charges or less) completely complexed siRNA at lower N:P ratios than

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longer polymers (80 charges or more).

In terms of media composition, it was previously


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reported that our ROMP-based PTDMs do not experience significant reduction in performance

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when tested in the presence of serum.20 Therefore, all cell data was collected in the presence of

3

serum.20

4

A summary of FITC-siRNA internalization efficiencies for homopolymer and block

5

copolymer PTDMs is shown in Figure 2, where Figure 2A presents the percentage of the cell

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population that received FITC-siRNA and Figure 2B presents the median fluorescence intensity

7

(MFI) of the cell populations. In both data sets, PTDM internalization efficiencies increased in a

8

charge-dependent manner up to 40 charges (dG20 and MePh5-b-dG20) and then diminish at

9

higher charge contents.

MePh5-b-dG20, with an MFI of 2,300, delivered the most amount of

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siRNA, which was three times larger than the next best PTDM, MePh5-b-dG10, with an MFI of

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800. This demonstrates that there is an optimal guanidinium content necessary for efficient

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internalization and above that charge content, the carriers are no longer as effective and show

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reduced performance.


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Figure 2. FITC-siRNA internalization in Jurkat T cells using homopolymer and block copolymer PTDMs. Jurkat T cells (cell density = 4x105 cells/mL) were treated with PTDM/FITC-siRNA complexes with an N:P ratio = 8:1 in complete medium for four hours at 37°C and compared to cells only receiving FITC-siRNA. All data was normalized to an untreated control. A) Percent FITC positive cells. B) Median fluorescence intensity (MFI) of the cell population. Data represents the mean ± SEM of three independent experiments. * p = < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant, as calculated by the unpaired twotailed student t-test. * represents significance between homopolymer and block copolymer PTDMs with the same charge content.

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From this data set, it can also be seen that the block copolymer PTDMs significantly

2

outperformed their homopolymer counterparts, particularly at charge contents of 40 or less.

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Both MePh5-b-dG20 and MePh5-b-dG10 had MFI values that were six times higher than their

4

corresponding homopolymers (dG20 and dG10, respectively). At larger charge contents, MFI

5

values were similarly diminished for both homopolymer and block copolymer PTDMs. The

6

block copolymer PTDM with 80 charges (MePh5-b-dG40) delivered FITC-siRNA to 66% of the

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cell population, which is approximately double the cell population that its homopolymer PTDM

8

counterpart, dG40, could reach. The observation that block copolymers outperformed their

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homopolymer counterparts is consistent with other reported guanidinium-rich siRNA transport

10

molecules.39,48

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supporting information (Figure S23). PTDMs were also tested for their ability to deliver FITC-

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siRNA into HeLa cells to demonstrate that they enabled internalization in adherent cells and to

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establish trends in a second cell type. Block copolymer PTDMs containing 20 and 40 charges

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(MePh5-b-dG10 and MePh5-b-dG20) were shown to deliver double the amount of siRNA than

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their homopolymer counterparts. PTDMs with 80 charges (dG40 and MePh5-b-dG40) were able

16

to facilitate the internalization of the most siRNA, with similar MFIs of 4,100 and 4,500,

17

respectively. A significant drop-off in internalization efficiency for the largest charged segment

18

was still observed (Figures S33-S36).

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viability using 7-AAD/Annexin V staining (Figure S26-S27 and Figures S37-38).

In addition to Figure 2, flow cytometry histograms can be found in the

All polymer-treated cells showed greater than 90%

20

In a separate viability study, promising PTDM/siRNA complexes were compared to

21

samples that were treated with PTDMs alone in the same working concentrations used for

22

complex formation. Cells were harvested four hours after treatment and washed. Samples were

23

split and viability was determined on one half of the cells. The other half were re-plated in fresh


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Biomacromolecules

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serum-containing medium and viability was assessed after an additional 24 hours of incubation.

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This not only assessed PTDM toxicity at the maximum free concentrations, but it also examined

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longer-term toxicity effects of the treatment. All cells exhibited greater than 90% viability as

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assessed by 7-AAD/Annexin V staining both at the four-hour and 24-hour time points (Figures

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S29-S31).

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proliferation. All cell populations were found to approximately double over the 24-hour period,

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suggesting that PTDM/siRNA treatment did not impair cell growth (Figure S32).

In addition, cells were also counted at the 24-hour time point to measure cell

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Based on these results, two block copolymer PTDMs, MePh5-b-dG5 and MePh5-b-dG20,

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were compared to a range of commercial reagents commonly used for siRNA internalization and

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delivery, including R9 (peptide, Peptide2.0), DeliverX (peptide, Affymetrix), Xfect (polymer,

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ClonTech), N-Ter (peptide, Sigma Aldrich), and RNAiMAX (lipid, Life Technologies). In

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addition, JetPEI, a common polyethyleneimine-based pDNA delivery reagent, was included. All

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commercial reagents were used as directed by the vendor.

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internalization efficiencies for block copolymer PTDMs and commercial reagents is shown in

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Figure 3, where Figure 3A presents the percentage of the cell population that received FITC-

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siRNA and Figure 3B presents the MFI of the cell populations. MePh5-b-dG5 and MePh5-b-

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dG20 were both shown to facilitate siRNA internalization in a greater percentage of cells than the

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commercial reagents (Figure 3A). In addition, MePh5-b-dG20 resulted in a nearly 10 folder

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greater MFI than the commercial reagents, indicating it was able to deliver quantitatively more

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cargo inside the cells (Figure 3B, Figure S24). Viability was assessed using 7-AAD / Annexin V

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staining (Figure S28). All samples had greater than 90% viability, with the exception of samples

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treated with RNAiMAX, which had viabilities closer to 80%.

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superiority of our PTDMs for siRNA delivery compared to common commercial reagents and

A summary of FITC-siRNA

This data demonstrates the


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Biomacromolecules

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establishes these materials as viable delivery vehicles for hard-to-transfect T cell lines, for which

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options for efficient delivery are limited.

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Figure 3. Comparison of PTDM and commercial reagent FITC-siRNA internalization efficiencies in Jurkat T cells (cell density = 4x105 cells/mL). Cells treated with PTDM/FITCsiRNA complexes with an N:P ratio = 8:1 or with commercial reagent/FITC-siRNA complexes (used as directed) in complete medium for four hours at 37°C and compared to cells only receiving FITC-siRNA. All data was normalized to untreated controls. A) Percent FITC positive cells. B) Median fluorescence intensity (MFI) of the cell population. Data represents the mean ± SEM of three independent experiments. All PTDM data is statistically different from the commercial reagents (p

Development of Guanidinium-Rich Protein Mimics for Efficient siRNA

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