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Oct 30, 2012 - Jirina Zackova Suchanova , Jitka Neburkova , Hana Spanielova , Jitka .... Integrated nanotechnology platform for tumor-targeted multimo...
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MS2 Viruslike Particles: A Robust, Semisynthetic Targeted Drug Delivery Platform Francis A. Galaway† and Peter G. Stockley* Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, U.K. S Supporting Information *

ABSTRACT: We show that viruslike particles (VLPs) reassembled in vitro with the RNA bacteriophage MS2 coat protein and an RNA conjugate encompassing a siRNA and a known capsid assembly signal can be targeted to HeLa cells by covalent attachment of human transferrin. The siRNA VLPs protect their cargoes from nuclease, have a double-stranded conformation in the capsid and carry multiple drug and targeting ligands. The relative efficiency of VLP reassembly has been assessed, and conditions have been determined for larger scale production. Targeted VLPs have been purified away from unmodified VLPs for the first time allowing improved analysis of the effects of this synthetic virion system. The particles enter cells via receptor-mediated endocytosis and produce siRNA effects at low nanomolar concentrations. Although less effective than a commercial cationic lipid vector at siRNA delivery, the smaller amounts of internalized RNA with VLP delivery had an effect as good as if not better than the lipid transfection route. This implies that the siRNAs delivered by this route are more accessible to the siRNA pathway than identical RNAs delivered in complex lipid aggregates. The data suggest that the MS2 system continues to show many of the features that will be required to create an effective targeted drug delivery system. The fluorescence assays of siRNA effects described here will facilitate the combinatorial analysis of both future formulations and dosing regimes. KEYWORDS: siRNA delivery, nanoparticle targeting, MS2, virus assembly

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INTRODUCTION

a synthetic virion (SV) to indicate that it was not an infectious vector yet had many of the properties of natural viruses. The molecular details of the assembly pathway of bacteriophage MS2 are the most detailed for any ssRNA virus. It is a 26 nm diameter, T = 3 capsid composed of 180 identical copies of a small coat protein (CP) subunit 129 aa long, that associate as 90 noncovalent dimers (Figure 1). The icosahedral shell is formed by the accurate placement of two distinct dimer conformers; there are 30 copies of a symmetric C/C (purple) and 60 copies of an asymmetric A/B (green/ blue) version.9 They exhibit classical quasi-equivalence10 in that one polypeptide loop in each subunit connecting the F and G β-strands (FG-loop) alters its conformation in a symmetry related way. Uniquely in viruses of this type we understand the molecular basis of this switching mechanism, without which the correct size and symmetry of the particle could not be specified correctly during assembly. We have shown that binding of RNA stem-loops to the free CP2 switches the FG-loop conformations from C/C-like to A/B-like via a long-range allosteric effect that is not highly sequence-specific.11−14 Although there is a well-

Some of the most significant reasons for novel drug failures during clinical trials are related to their pharmacokinetics and bioavailability.1 The ideal of being able to maintain an effective drug concentration at the target molecule or tissue without offtarget side effects is a longstanding challenge.2,3 Modern biology has also thrown up some additional challenges in the form of potentially therapeutic macromolecules, ranging from peptides and proteins to oligonucleotides, whose sizes and physical properties are very different from the traditional small molecules developed by the Pharma industry.4−6 Many of the targets of these new drugs are intracellular, yet their properties prevent them being easily taken up into cells. In response to these challenges in the early 1990s, we developed a strategy for targeted drug delivery based on VLPs.7,8 Natural viruses encode systems for targeting only subsets of their host’s cells where they manage the considerable feat of internalizing their nucleic acids or nucleoprotein cores, which are macromolecules or complexes with molecular weights ranging into the millions of daltons. Our idea was to use these features as a template to create a semisynthetic drug delivery platform based around our knowledge of the assembly pathways of the RNA bacteriophage MS2. The goal is to produce an inert VLP chemically altered to (a) encapsidate a drug of choice and (b) display targeting molecules to make it bind and internalize into specific cells. We termed this platform © 2012 American Chemical Society

Special Issue: Viral Nanoparticles in Drug Delivery and Imaging Received: Revised: Accepted: Published: 59

June 19, 2012 October 25, 2012 October 30, 2012 October 30, 2012 dx.doi.org/10.1021/mp3003368 | Mol. Pharmaceutics 2013, 10, 59−68

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Figure 1. Components of the siRNA MS2 VLP. (A) External view and central cross-section seen along the 3-fold axis of the MS2 TR VLP (left) showing the RNA as a cartoon model and the coat protein dimers that comprise the protein shell as ribbons color-coded as purple (C/C dimers) and green/blue (A/B dimers). Enlarged views of the dimeric capsomeres are shown (right) with stick models for the surface lysine residues and for TR bound to the inner surface of the A/B dimer. A transferrin molecule is shown adjacent to the VLP as a ribbon cartoon for scale. A cartoon of the TRBcl2/ds construct together with its sequences and expected secondary structure are shown below. (B) A 15% (w/v) native polyacrylamide gel (left) illustrating the formation of the TRBcl2/ds: lanes 1, 10bp DNA ladder; 2, TRBcl2/ss, 40 nt; 3, silencing strand, 21 nt; and 4, TRBcl2/ds. A similar gel showing the products of digestion with Dicer is shown on the right viewed under several different wavelengths: lanes 1, 10bp DNA ladder; 2, TRBcl2/ds; and 3, TRBcl2/ds plus Dicer.

and antisense oligonucleotides.7,17,18 The assembled material is easily purified from the starting materials and assembly intermediates. It is possible to decorate the external surface with various targeting ligands, which to date have included covalently attached human transferrin, anti-HIV gp120, human CD4, as well as anti-MS2 IgGs. We have shown that both targeting systems and drugs are able to function selectively in cell culture, entering cells via receptor-mediated endocytosis (RME), as expected. Entry into the endosomal pathway is useful because the progressive lowering of the pH along this pathway leads to disassembly of the VLP and escape into the endosome of the druglike entity. How these cargoes further escape the endosomal membrane into the cytoplasm is currently unknown, but there are precedents, albeit for animal viruses, for the endosomal entry of ssRNA viruses that pass their genomes into the cytoplasm in a process that is resistant to nuclease.19 Here we describe the use of the MS2 SV VLP for targeted delivery of a siRNA. We have carefully monitored the

known high affinity CP-binding site (TR) within the viral RNA, it appears that multiple such packaging signals have evolved within the genome to make assembly efficiency in vivo both rapid and highly accurate.15 Understanding the natural assembly process is useful in creating the targeted VLPs because it underpins our strategy for their production. In vitro reassembly is used for this purpose. This is possible because the wild-type virion can be disassembled by treatment with acetic acid and then reassembled in the presence of a variety of oligonucleotides by simply raising the pH toward neutrality.12,16 The scale of these reactions can easily be increased using recombinant CP expressed in Escherichia coli in the absence of phage RNA, which results in the formation of very high yields of MS2 T = 3 VLPs8. The efficiency and fidelity of these in vitro reactions is lower than those of an in vivo assembly reaction, but these effects can be minimized. We have used a number of chemically synthesized TR variants to trigger capsid reassembly in this system, including TRs derivatized with ricin A chain (RAC) 60

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with only annexin V or only propidium iodide. Untreated cells were exposed to ultraviolet radiation for 5 min. These cells were harvested and stained with propidium iodide as a necrotic control. For an apoptotic control, cells were treated with cisplatin (final concentration 5 μM in the medium) 24 h prior to flow cytometry. These cells were harvested and stained with annexin V. A well of cells heavily transfected with RNA (HiPerfect; 100 nM final concentration of RNA) was harvested and prepared as described above, but was stained with neither annexin V nor propidium iodide. Intracellular Antibody Staining. The cell growth medium was collected and retained. The cells were washed with DPBS twice and then harvested with TrypLE Express at 37 °C. The harvested cells were collected with the original growth medium and then centrifuged at 1000 RCF for 5 min. The cell pellet was resuspended in paraformaldehyde (1% in DPBS) for 5 min at room temperature. The fixed cells were centrifuged at 1500 RCF for 5 min, resuspended in DPBS and centrifuged again at 1500 RCF for 5 min. The cell suspension was then transferred to a clean tube and incubated in permeabilization buffer for 20 min on ice. The anti-Bcl2 antibody (FITC labeled) was added (20 μL per 106 cells). The cells were incubated with the antibody for 1 h on ice and centrifuged at 1500 RCF for 5 min before resuspending in DPBS. The antibody stained cells were kept on ice prior to flow cytometry analysis. An isotype control antibody (FITC labeled) was used as described above to stain untreated cells for a background fluorescence control. Fluorescence Activated Cell Sorting of Transfected Mammalian Cells. The flow cytometry analysis of cells was performed on a FACSAria II (Becton Dickinson) or a FACSCalibur (Becton Dickinson). The 488 nm laser was used with a 525/10 detector (FITC) and a 695/20 detector (PI). The 633 nm laser was used with a 660/10 detector (Cy5). The compensation was set using fluorescent beads (Becton Dickinson) according to the manufacturer’s instructions. The initial gate was set to distinguish cell events from non-cell events using the forward and side scatter (FSC and SSC) of untreated cells. This gate had low stringency because many of the experimental treatments produced distinct morphological changes from “healthy” cells. The compensation parameters were further refined using the staining controls. The annexin V stained cells (untreated and cisplatin) were used to adjust the 696/20 detector signal. The propidium iodide controls (untreated and ultraviolet radiation) were used to adjust the 525/10 and 695/10 detector signals. The cyanine 5 control (high level RNA transfection) was used to adjust the 695/20 detector signal. In a typical experiment only the 525/10 detector signal needed to be adjusted for the propidium iodide bleed-through. The second gate was set to distinguish nontransfected and transfected cells. The untreated cells were used to set the upper cyanine 5 fluorescence intensity for nontransfected cells (more than 99% of events). The third gate was a quadrant designed to sort cells into one of four populations (healthy, early apoptosis, late apoptosis, or necrotic). The cells treated with cisplatin and stained with annexin V were used to set the upper propidium iodide fluorescence intensity for healthy cells. The cells exposed to ultraviolet radiation and stained with propidium iodide were used to set the upper annexin V fluorescence intensity for healthy cells. The cells stained with an anti-Bcl2 antibody were not sorted. For each sample, 104 cell events were collected for flow cytometry analysis. The entire sample population was sorted for

construction and properties of the TR−siRNA conjugate, its reassembly efficiency, and the covalent decoration of a targeting transferrin. Potent and specific RNA silencing was achieved by this route. We also discuss the potential of this system for combinatorial drug delivery and the ways in which the efficacy of each formulation could be monitored.

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EXPERIMENTAL SECTION RNA and Protein Methods. The production and characterization of synthetic RNA oligonucleotides,20 and their structural analysis, together with the reassembly of VLPs and their subsequent derivatization with holo-transferrin as a targeting ligand followed previous literature precedents except where indicated in the text. The details of these protocols are described in the Supporting Information. Cell-Based Assays. Mammalian Cell Transfection. The cells were grown in vitro to ∼90% confluence and harvested using TrypLE Express (a recombinant trypsin; Gibco). The cells were diluted to 0.5 × 105 cells·mL−1 in medium (10% FBS). Then 2.4 mL of these diluted cells were seeded in each well of a 6 well plate. The cells were returned to the incubator (5% CO2, 37 °C, humidified). The RNA (whether free or in VLPs) was diluted in serum-free medium to a total volume of either 94 μL (for lipid-based transfection) or 100 μL (VLPs). For lipid-based transfection, 6 μL of HiPerfect (Qiagen) was added to the free RNA to give a total volume of 100 μL. This was vortexed and left for 10 min at room temperature. The RNA concentration in the 100 μL was 25× the final concentration. The RNA concentration was calculated from the dye absorbance (sense strand, no dye; antisense strand, Cy5). The transfection formulations were then added dropwise to the recently seeded cells (final volume 2.5 mL and 1× RNA concentration). This is the 0 h time point. The well plates were agitated and returned to an incubator for 24−72 h prior to analysis by flow cytometry. The controls for each flow cytometry analysis of transfection were untreated cells and cells incubated with free RNA (no transfection formulation). Two other control experiments were performed. In addition to treatment with RNA formulations (HiPerfect or VLPs), human holo-transferrin was added to a final concentration of 100 nM at 0 h. Prior to transfection with RNA, the cells were transfected with a siRNA against the transferrin receptor. The cells were seeded in wells as described above. The transferrin receptor siRNA was prepared as described above, using HiPerfect (Qiagen). The final concentration of this siRNA was 50 nM in a well. The transfection with the test subject RNA was then carried out as described above, but 48 h after the original cell seeding. This second transfection step is referred to as 0 h (not the transferrin receptor siRNA transfection 48 h beforehand). Apoptosis Assay. The cell growth medium was collected and retained. The cells were washed with DPBS twice and then harvested with TrypLE Express at 37 °C. The harvested cells were collected with the original growth medium and then centrifuged at 1000 RCF for 5 min. The cell pellet was then washed twice with DPBS and once with annexin binding buffer. The cells were resuspended in 1 mL of annexin binding buffer containing annexin V (2 μg·mL−1) and propidium iodide (4 μg·mL−1). The stained cells were kept on ice for 15−30 min prior to analysis by flow cytometry. Flow cytometry controls were prepared for the apoptosis assay using cells grown in parallel with the experimental treatments. Untreated cells harvested from wells were stained 61

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may represent dissociated antisense strands. However, the assay does suggest that the TRBcl2/ds should have the expected siRNA function. Reassembly and Properties of MS2 VLPs Containing the siRNA Conjugate. In principle the ideal ratio for capsid assembly in the MS2 system is a TR:CP2 molar ratio of 0.66:1, since TR switches the coat protein dimer conformation required for each of the 60 A/B dimers within a T = 3 capsid. In practice we find that a range of TR:CP2 ratios support assembly in vitro since the RNA binding acts by extending the lifetime of the A/B conformer, which is nevertheless infrequently accessible to the unliganded protein, allowing assembly to occur when not all A/B dimers are actively switched by TR. The situation to avoid is one in which all the coat protein dimers are saturated with TR, in which case little assembly occurs because the complex is kinetically trapped in the absence of RNA-free species.12−14 There is a balance to be struck between the number of drug species that can be encapsidated and the efficiency of the reassembly process. For short RNA oligos like TR, it is possible to soak crystals of capsids directly, and many crystal structures of these complexes have been determined in which the occupancy of the RNA is greater than 60/capsid, i.e., at high concentrations TR can bind C/C like dimers in a capsid, but this does not cause conformational change, presumably because that would disrupt the capsid and is therefore disfavored.24,25 We therefore usually perform a titration at differing TR:CP2 ratios (Figure 2). In this case all ratios showed reassembly of a T = 3 capsid, as judged by TEM, although this was incomplete for ratios below 1:50. The TRBcl2/ds conjugate carries additional flanking sequences adjacent to TR, and these have been shown to partially relieve the kinetic trap seen for TR.14 The protein dependency of the assembly reaction is consistent with the observed assembly efficiency. At the higher ratios species migrating more slowly than the T = 3 capsid are also seen. Often these are aggregates, e.g., the rods seen in Figure 1B. We therefore chose a reassembly ratio of 1:10 TRBcl2/ds:CP2 for larger scale production of the siRNA VLP, since this gave the highest yield of T = 3 capsid for the least amount of aggregate. RNase A digestion of the reassembled products at a ratio of 1:1 showed that the VLP protected its RNA cargo from digestion while the faster moving species, corresponding to assembly intermediates, were degraded (not shown). The conformation of TRBcl2/ds inside the VLP was then probed using cleavage with lead ions which can penetrate the center of the particles along the pores at the capsid vertices (Figure 2B). The rate of hydrolysis by lead ions of individual RNA phosphodiesters is determined by their secondary structures. Double stranded regions cleave more slowly.26 In order to confirm that the antisense strand remained annealed when TR was bound to the protein shell, we reassembled both TRBcl2/ ds and TRBcl2/ss RNAs at a 1:10 ratio for 72 h. Under these conditions both samples form T = 3 capsids with good yield. These were then incubated with lead(II) acetate at room temperature for 30 min, before the reactions were terminated by phenol−chloroform extraction and the cleavage products analyzed in a denaturing gel (Figure 2C). The unencapsidated TRBcl2/ss shows lead ion cleavage at every nucleotide position including within the TR region, presumably because there is a conformational equilibrium allowing the lead access to a single strand conformer.27 In contrast the free TRBcl2/ds is much more resistant to cleavage throughout its length, consistent with it adopting the secondary structure shown in Figure 1.

downstream PCR and Western blotting analysis. The sorted cells were collected in LiDS lysis buffer (2×) and stored at −80 °C. Flow Cytometry Data Analysis. Mammalian cell transfections, apoptosis assays, and antibody staining were analyzed in Cyflogic v1.2.1. A maximum of 10000 events were plotted. Compensation and gates were applied as described above using control samples. The number of events in each gate and the mean fluorescent intensity values for each cell population were exported to Excel 2007 for graph plotting and error bar calculations. VLP Internalization Analysis. HeLa cells were seeded on glass coverslips (no. 1.5) in a 24 well plate and grown to 50− 70% confluence. The growth medium was removed, and the cells were washed with room temperature medium. The cells were then stored at 8 °C for 20 min. Then VLPs were added with a final RNA concentration of 25 nM (as measured by Cy5 absorption). The HeLa cells were incubated with the VLPs at 8 °C for 1 h. The cells were then washed three times with chilled medium (8 °C). Warm medium (37 °C) was added, and the cells were returned to 37 °C. This was the 0 min time point. After 0, 5, and 30 min the cell medium was replaced with paraformaldehyde (1% in DPBS) for 15 min. The fixed cells were washed with DPBST twice. The cells were then incubated with DAPI (2 μg·mL−1) for 10 min. The coverslips were mounted on glass slides using Fluoromount-G (Southern Biotech) and allowed to dry. The slides were observed on a Delta Vision deconvolution microscope (Olympus). A 60× magnification objective lens (oil) was used to acquire images. The exposure was adjusted using nonstained control slides. SoftWoRx image acquisition and analysis software was used to acquire a z stack of images and perform a deconvolution analysis.

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RESULTS Design and Production of a siRNA−TR Conjugate. For the test siRNA we chose the anti-Bcl2 siRNA sequence used previously by McNamara,21 who showed that it could be coupled with a targeting aptamer to enable it to be taken up into cells via RME, both in culture and in vivo in mice. Their aptamer-siRNA construct appeared to be capable of escaping the endosome following internalization and was a better substrate for Dicer than expected of a 21 nt ssRNA. This sequence was most effective when the siRNA region was annealed to its antisense complement. The sequences of the TR−siRNA conjugate and antisense oligos used here are shown in Figure 1A. Cy3 and Cy5 dyes were added to the 5′ ends of each oligo, either by direct addition as a phosphoramidite (Cy3) or by postsynthesis chemical modification of the 5′ terminal nucleotide (Cy5), to facilitate studying their cellular fates. These oligos were synthesized and purified using our standard protocols20,22and their identities confirmed by mass spectrometry. Then the purified single strand oligos were annealed and the dsRNA product gel was purified. The functionality of TRBcl2/ds was then tested in vitro using recombinant Dicer (Figure 1B). If the construct is a Dicer substrate, then the TRBcl2/ds would be expected to enter the RNAi pathway.23 The TRBcl2/ds band intensity decreased following Dicer incubation, and three new bands appeared. Two of these correspond to the expected double-stranded ∼21 nucleotide species (the anti-Bcl2 siRNA, Cy5 labeled) and the TR fragment (Cy3 labeled). The third species is Cy5 labeled and migrates faster than the 20 bp DNA marker, suggesting it 62

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Interestingly the cleavage patterns are significantly different for RNAs that have been encapsidated, showing significant base specificity of cleavage. The TRBcl2/ss is cleaved at several sites within the siRNA region adjacent to disruptions of fully base paired sections (see Figure 2C) while these sites are protected for TRBcl2/ds consistent with them being in the desired duplex structure. Purification and Covalent Attachment of Transferrin to siRNA VLPs. The siRNA VLP product formed in 1:10 RNA:CP2 reaction was chosen for the drug delivery studies. Covalent attachment of a targeting ligand is the next step in SV formulation, and we chose to use human transferrin (HTfn) for this purpose since it is readily available commercially, has a very well characterized internalization mechanism, and had been used successfully by us previously.7 The cross-linking chemistry was also the same as described previously, using standard amine-to-sulfhydryl chemistry (see Supporting Information). Protected thiol groups were introduced on the VLP and maleimide groups onto the apo-form of HTfn, both via surface lysine side chains. This was followed by the deprotection of the VLP and then cross-linking with maleimide HTfn. Subsequent characterization and drug delivery studies require larger scale preparations than the analytical reactions illustrated in Figures 1 and 2. We therefore examined the ease of scale up by a factor of 200, by doubling the concentration of the CP2 (to 40 μM) and the volume (from 10 to 1000 μL). Under these conditions capsid assembly was either inefficient or not detectable. Two parameters needed to be altered to achieve larger scale reassembly. The dissociated coat protein is purified in 20 mM acetic acid and the 40 mM ammonium acetate reassembly buffer is easily overwhelmed if there is any carry over from the glacial acetic acid used in virion or recombinant VLP dissociations. The reassembly buffer was therefore changed to 100 mM Tris-HCl, pH7. In addition it was necessary to reduce the volumes of the reassembly reactions from 1 mL to 100 μL. With these alterations the reactions were reproducible and in principle could be automated for production of much larger samples. The reassembled siRNA VLP was purified from assembly intermediates and aggregates by centrifugation through a sucrose density gradient (Figure 3). Analysis of the major peak showed it to be T = 3 VLPs containing the TRBcl2/ds as expected. Quantitation of these reactions suggested that reassembly yield was 80 ± 10% with respect to the starting concentrations of CP2 and TRBcl2/ds. The purified VLPs were then derivatized with HTfn7 (Figure 3). The cross-linked products were analyzed on a 3D field gel filtration system that allows collection of the spectrum between 200 and 700 nm every 0.5 s. This permits refined analysis of composition and size of the components eluting from the column (see Supplementary Figure 1 in the Supporting Information and Table 1). The addition of Fe3+ to apotransferrin was detected through the emergence of an absorbance peak at 465 nm. The A465 was used to identify holo-transferrin in the protein mixture and hence VLPs that were labeled with transferrin, i.e., the SV fraction. The 3D field size exclusion chromatography also allowed the detection of aggregates via their light scattering properties, and provides simultaneous quantification of the amounts of TRBcl2/ds within the capsid. A linear sucrose density gradient was chosen to purify the VLP because this removed aggregates, accounting for the reduction in apparent hydrodynamic radius (Table 1). During the coupling reaction the mean TRBcl2/ds content of

Figure 2. VLP assembly and RNA protection. (A) A native agarose gel and negative stain TEMs of VLP reassembly products in the presence of TRBcl2/ds: lanes 1, 10bp DNA ladder, stained with ethidium bromide; 2, TRBcl2/ss; 3, silencing strand (not Cy3 labeled); (4) TRBcl2/ds; VLP reassemblies at 1:1 (5); 1:2 (6); 1:10 (7); and 1:50 (8) molar ratios (TRBcl2/ds to coat protein dimer). TEM scale bars indicate 100 nm. (B) Lead acetate structure probing of encapsidated TRBcl2 conjugates. 1:10 molar ratio assembly reactions using TRBcl2/ ss or TRBcl2/ds in separate reactions were allowed to proceed for 72 h at 4 °C (forming VLP(TRBcl2/ss) and VLP(TRBcl2/ds), respectively). The samples were incubated with lead(II) acetate for 30 min and phenol−chloroform extracted, and the RNA was analyzed on a 20% (w/v) denaturing gel: lanes 1, 4, 7, and 10 and 2, 5, 8, and 11 contained 1.0 or 0.1 mM Pb(II) respectively. The nucleotides are numbered by presuming that the top band represents the full length TRBcl2/ss (40 nt) and that there was cleavage at every position within the free TRBcl2/ss (lanes 10 and 11). Presumed secondary structures for the encapsidated oligos are shown above with the most frequent cleavage marked in red. 63

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Table 1. Summary of 3D Field Chromatography Analysisa diameter (nm) VLP (E. coli) VLP (TR) VLP (TRBcl2/ds) VLP (TRBcl2/ds), purified transferrin SV (TRBcl2/ds)

RNA molecules/ capsid

transferrin molecules/capsid

26 25 28 25

n/a 4.5 ± 0.4 17.2 ± 0.5 7.5 ± 0.9

n/a n/a n/a n/a

7.6 34

n/a 3.5 ± 0.6

n/a 15.2 ± 2.3

a

Apparent VLP sizes and TRBcl2/ds content (see Supplementary Figure 1 in the Supporting Information).

transfection reagent (HiPerfect, Qiagen; referred to here as Commercial) and the nontargeted VLP. The procedure for the Commercial reagent use was optimized as per the manufacturer’s instructions. When HeLa cells were preincubated with the SV at 4 °C prior to observation by deconvolution microscopy, the Cy5 signal of the TRBcl2/ds localized to the cell exterior (Figure 4A). Upon raising of the temperature to 37 °C the SV(TRBcl2/ds)but not the undecorated VLP(TRBcl2/ds)was internalized within 30 min, consistent with previous colocalization studies.17 Flow cytometry was used to measure the extent of intracellular TRBcl2/ds delivery for samples of 10000 cells (Figure 4B). HeLa cells with a Cy5 signal greater than 99.0% of an untreated sample were classed as “transfected”, meaning that the TRBcl2/ds had been introduced into the cell. (The location of TRBcl2/ds in these cells was not determined, but its persistence beyond 48 h in HeLa cells suggests that the TRBcl2/ds could escape the endosome). The ability of the SV, Commercial reagent, and VLP to transfect cells was compared over 72 h for an initial TRBcl2/ds concentration of 5 nM in the culture medium (Figure 4C). At 6 h the percentage of transfected cells was 71% (Commercial), 63% (SV), and 53% (VLP). At 48 h both the Commercial reagent and SV achieved greater than 90% transfection, a value that may indicate exposure of 100% of the cells given the detection sensitivity, compared to 66% for the VLP. The percentage of transfected cells increased from 6 to 24 h, but the intracellular TRBcl2/ds concentration decreased due to cell growth and division. The depletion of space and nutrients slowed growth after 24 h while the toxicity of the treatment reduced cell numbers after 48 h. These results are entirely consistent with an RME entry route, but to test this assumption directly a commercial siRNA (Qiagen) was used to reduce transferrin receptor expression in the test cells prior to addition of the SV. Holo-transferrin was also added to the culture medium as a direct inhibitor for SVHTfn receptor interaction (Figure 4C). These controls reduced the SV transfection properties to those of the VLP (from 64% to 53% at 6 h and from 90% to 66% at 48 h). The VLP and Commercial reagent were unaffected by these controls. The transferrin-targeted uptake of the SV had enhanced its ability to deliver a siRNA compared to the VLP. However, there was a background uptake associated with the VLP that occurred independently of the RME pathway, and the extent of endosomal escape by the TRBcl2/ds was not determined. Quantitation of siRNA Effects Delivered by VLPs. The target-specific effect of TRBcl2/ds was assessed by measuring Bcl2 expression and cytotoxicity in transfected HeLa cells using flow cytometry (Figure 5). The HeLa cells were incubated with the SV, Commercial reagent, or VLP for 48 h and then stained with an anti-Bcl2 antibody or annexin-V and propidium iodide.

Figure 3. Covalent decoration of siRNA VLPs with human transferrin. (A) VLP purification. Reassembly reactions (4 °C for 72 h) were loaded on a linear sucrose density gradient (15−45% w/v). Samples were fractionated using a piston gradient fractionator with a UV detector set at A260. The major peak was collected and analyzed by TEM (shown below). (B) The purification of SVs from the transferrin/VLP cross-linking reaction. The cross-linking reaction components were separated by size exclusion chromatography (Superose 6) and the peak fractions collected. The A465 indicates transferrin-bound Fe3+ and was used to identify the SV fraction (highlighted in gray). The other peaks eluting were assigned as excess transferrin (18 mL elution volume) and residual cross-linking reagents and starting materials. (C) The peak fraction samples from (B) were analyzed by TEM, SDS−PAGE, and Western blotting with antibodies to MS2 coat protein and human transferrin. Gel lanes are (1) transferrin, 77 kDa; (2) SV fraction; and (3) recombinant MS2 coat protein. * indicates the SV species detectable by both MS2 and transferrin antibodies. TEM scale bars indicate 100 nm.

each capsid was reduced from 7.5 (VLP) to 3.5 (SV). The estimated number of transferrin molecules per SV was 15.2. Cellular Delivery of siRNA by Synthetic Virions. The silencing sequence in TRBcl2/ds has previously been shown to be effective at 5 nM in the culture medium of HeLa cells when targeted for RME by an aptamer.21 The ability of the SV to deliver the siRNA was tested using adherent HeLa cells. The efficiency of SV uptake was compared to a commercial lipid 64

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Figure 4. Internalization of synthetic virions by HeLa cells. (A) Fluorescence microscopy observation of SV(TRBcl2/ds) uptake by HeLa cells. HeLa cells were incubated with synthetic virions (25 nM, TRBcl2/ds) at 4 °C for 30 min to allow surface binding but not internalization. The cells were then incubated at 37 °C for 5 and 30 min prior to fixation and image acquisition using deconvolution microscopy. DAPI staining (nuclei) is depicted in blue, and Cy5 emission (TRBcl2/ds) is depicted in red. Bars = 20 μm. (B) Time-dependent transfection of HeLa cells using synthetic virions. HeLa cells were incubated with synthetic virions (TRBcl2/ds at 5 nM in the total growth medium) for 0, 6, and 72 h. The cells were harvested and observed with flow cytometry (upper panel). The gate set using controls was used to designate transfected cells (purple box). (Left) Percentage of cells transfected (bars are absolute error of 3 replicates). (Right) Mean fluorescent intensity of the whole population (bars are standard deviation across 104 cells). (C) SV(TRBcl2/ds) uptake compared to a commercial lipid transfection reagent and the VLP(TRBcl2/ds). HeLa cells were incubated with TRBcl2/ds using a commercial lipid reagent (HiPerfect), VLPs, or SVs (TRBcl2/ds at 5 nM in the total growth medium for all formulations). HeLa cells were also treated 24 h earlier with a transferrin receptor (TFRC) siRNA (Qiagen) and then incubated with synthetic virions for 6−24 h (Δ). In addition HeLa cells were incubated with synthetic virions in the presence of transferrin (100 nM in the total growth medium) for 48 h (×). The cells were harvested and observed with flow cytometry. (Left) Graph showing the percentage of cells transfected over time (absolute error of 3 replicates). (Right) Mean fluorescent intensity of the whole cell population over time (standard deviation across 104 cells).

iodide to identify cells as apoptotic or necrotic, respectively (Figure 5B). (Cells may quickly progress into necrosis and so not be counted by an apoptotic stain alone.) In each sample the nontransfected cells were used to normalize the transfected population, giving an estimate of the toxicity attributable to intracellular TRBcl2/ds exposure (Figure 5B). For a TRBcl2/ds concentration of 5 nM, the SV killed ∼25% of HeLa cells, the Commercial reagent ∼20%, and the VLP less than 5%. For a 25 nM treatment, the SV killed ∼40%, the Commercial reagent ∼45%, and the VLP less than 5%. The addition of holotransferrin reduced the SV toxicity to ∼15%. The reduction in Bcl2 expression with VLP treatment was not sufficient to induce apoptosis at 48 h whereas the SV was as effective as the Commercial reagent. The use of a scrambled TRBcl2/ds sequence did not reduce Bcl2 expression, although it did result in some (∼7%) necrosis at a 25 nM dose of SV (Supplementary Figure 2 in the Supporting Information). The functionalization of the VLP surface with transferrin may be responsible for some of the necrosis at higher TRBcl2/ds doses. An RT-PCR analysis of cells sorted using flow cytometry

RNAi effects typically take 24−72 h to manifest. The cell uptake results above suggest that >60% of cells were transfected after 6 h, so a 48 h time point was chosen to assay the extent of Bcl2 expression and apoptosis. This avoids issues with reagent dilution and cell death at later time points. The Commercial reagent was expected to produce the maximum possible Bcl2 knockdown and cytotoxicity for the experimental conditions given its level of siRNA internalization. At an initial TRBcl2/ds concentration in the culture medium of 5 nM, both the SV and Commercial reagent reduced Bcl2 expression by ∼55% when measured by antibody staining (Figure 5A). The VLP at this concentration reduced Bcl2 expression by ∼30%. At 25 nM, the Bcl2 expression was reduced by ∼90% (Commercial reagent), ∼75% (SV), and ∼55% (VLP). The addition of holo-transferrin to the medium reduced this SV knockdown effect selectively to ∼35%. The Bcl2-specific effect of TRBcl2/ds was enhanced by the SV compared to the VLP and was equivalent to that of the Commercial reagent at the 5 nM dose. The cytotoxicity of Bcl2 knockdown was assessed by using annexin-V and propidium 65

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Figure 5. SV(TRBcl2/ds) effect on Bcl2 expression and induction of apoptosis. (A) Reduction in Bcl2 protein expression. HeLa cells were incubated with TRBcl2/ds using a commercial reagent (HiPerfect), VLPs, or SVs for 48 h. The TRBcl2/ds concentration was 5 or 25 nM in the total growth medium for each formulation. In addition HeLa cells were incubated with SVs in the presence of transferrin (100 nM in the total growth medium). The cells were harvested, stained with a Bcl2 antibody, and observed with flow cytometry (upper panel). The maximal (green oval) and minimal (purple oval) expression of Bcl2 were estimated using untreated cells and an isotype control antibody. (Bars are standard deviation across 104 cells.) (B) The toxicity of HeLa cell treatment with synthetic virions. HeLa cells were incubated with TRBcl2/ds using a commercial reagent (HiPerfect), VLPs, or SVs for 48 h. The TRBcl2/ds concentration was 5 or 25 nM in the total growth medium for each formulation. In addition HeLa cells were incubated with SVs in the presence of transferrin (100 nM in the total growth medium). The cells were harvested, stained with annexin V and propidium iodide, and then observed with flow cytometry (upper panel). The controls were used to designate early apoptosis cells (green box) and necrotic cells (red box). The region of overlap in staining was used to designate late apoptosis cells. The HeLa cells were sorted using the Cy5 fluorescence emission into a nontransfected and a transfected population. The toxicity of transfection was normalized using the nontransfected population for comparison between the different treatments (lower panel).

found a ∼90% reduction in Bcl2 mRNA expression in the apoptotic population following TRBcl2/ds treatment (Supplementary Figure 3 in the Supporting Information). This effect was Dicer-dependent. The Bcl2 mRNA expression in a population of transfected, but annexin negative, HeLa cells was double that of the control, suggesting a selective pressure from the toxic treatment. Long RNA sequences and repetitive protein structures may conceivably trigger innate immune responses and hence toxicity through cytokine signaling. In this instance no innate immune response to the TRBcl2/ds formulations could be identified (Supplementary Figure 4 in the Supporting Information), eliminating this as a source of toxicity in the HeLa cells, but future in vivo studies will need to consider this further. In summary, the SV-mediated delivery of TRBcl2/ds caused Bcl2 knockdown and apoptosis in HeLa cells as effectively as a commercial lipid transfection reagent. The transferrin-targeted uptake of the SV was responsible for the enhanced targetspecific effect of TRBcl2/ds compared to the VLP.

this route showed a time-dependent loss of both bcl transcripts and their translation product. The cells showed a clear cytotoxicity entering the apoptotic pathway as determined using standard phenotype screens. Since the siRNA mechanism requires the RNA conjugates to be in the cytoplasm, the results suggest that, as with other cargoes we have delivered via this system including RAC, the “drug” has escaped from the late endosome. Escape from the SV itself is presumably due to low pH-mediated disassembly, but the exact mechanism of penetration through the endosomal membrane remains elusive and may limit efficacy. In this study our estimates of SV doses received by the target cells were improved by use of the UV absorption at 465 nm of holo-transferrin, which in combination with a more sophisticated chromatography system permitted isolation of only VLPs carrying the charged targeting ligand. As with our other exemplifications of this technology the use of siRNA SVs yields effective doses in the nanomolar range. We have also explored ways to self-assemble SVs on a much larger scale, which will be needed if this technology is to advance into animal trials (see below). We have also developed assay systems based on fluorescence detection that are readily adapted to higher throughput formats that would allow assay of the results of such trials. For instance here we found that commercial cationic lipid systems initially deliver much more siRNA into cells than the SV but that the SV route yielded a higher degree of apoptosis, implying that its cargo more easily enters the RNAi pathway. In addition we showed that, somewhat at variance to our earlier studies7 there was some uptake of cargo via an unmodified VLP

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DISCUSSION Here we have further demonstrated the versatility of the MS2 SV system in directed drug delivery by adapting previous formulations7,17,18 to target a siRNA against a known tumor target, the bcl oncogene transcript. Use of human holotransferrin covalent conjugates directs these nanoparticles into the RME pathway of HeLa cells. Controls against both the functional receptor and its expression confirm that this uptake is mediated by the Tfn receptor. Cells taking up the siRNA via 66

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properties. The Francis group has demonstrated more extensive chemical modification of the capsid, including the addition of nucleic acid aptamers to the outer surface.38,39 Finn’s group has begun to use in vivo assembly of phage VLPs, especially with Qβ, to assemble an interesting array of reagents.40,41 Peabody’s group has recently recapitulated our earlier work on a variety of cargoes and also shown that MS2 SVs can deliver siRNAs successfully.42 Their use of multiple peptide targeting and release conjugates at the surface further exemplifies our earlier suggestions.43 Most recently the Wang group demonstrated that MS2 SVs could be used to encapsidate and deliver miRNA precursors and to deliver these efficiently into cells following covalent attachment of HIV Tat protein.44 They also showed that the SVs had little toxicity in mice. These developments suggest that the exploitation of these systems is being actively explored, and we await further developments with interest.

and that this internalization route appeared independent of the Tfn-receptor dependent entry of the SVs. Since uptake of the cargo via a SV gave a greater RNAi-mediated effect than with the unmodified VLP, it seems likely that the uptake route is important for entry into the RNAi pathway. Cationic lipids form large invaginated bilayers that appear to physically trap their RNA cargoes, and it is not clear how they would subsequently release them. The architecture of these lipid− RNA mixtures can be controlled by altering the formulation of the lipids, and this has led to partial targeting to tissues based on physical properties.28,29 Such reagents are in clinical trials for anticancer applications.30 However, the precise numbers and cellular locations of the RISC complexes within the target cells are still unclear.31 The total numbers appear to be fairly low, so, even though they provide amplification of the input siRNA signals, it is highly likely that such systems could be overwhelmed by delivery of very large amounts of substrate, resulting in off-pathway and other side effects. Sorting out the complexities of such issues before animal trial experiments will require extensive and systematic analysis of dosing regimes and time courses, as well as extensive variation in targeting and cargo formulations. These are exactly our goals for the higher throughput assays envisaged. Since we introduced the SV concept, several other groups have begun to develop drug and imaging reagent delivery systems based around VLPs, most notably the use of CPMV.32−34 Such systems have a number of potential advantages. These include ease of manufacture, the cost of goods, and their well-characterized molecular architectures. They also exploit the basic molecular recognition strategies of natural viruses which have obviously proven to be successful in the exacting world of biological evolution. The multidentate nature of targeting ligands and the very large cargo capacity of VLPs are other features that recommend them for this type of application. At the time of our initial work a number of disadvantages to this approach were also identified. First there was a question of which “drugs” or other cargoes needed this type of delivery. The advent of RNA silencing and the identification of a plethora of intracellular potential drug targets, as well as the advances in medical imaging technology, have largely provided answers to these questions. Then there is the issue of using a protein reagent and the likely immune responses that can be expected. This is indeed a likely problem, but strategies have been developed to overcome it, e.g., by PEGylation, and we are developing formulations to reduce this effect. For instance, increasing the surface coverage of the MS2 particle with targeting ligands masks the underlying viral epitopes.8,18 In addition the surface attachment of nucleic acid aptamers that recognize cell surface markers is likely to have the double benefit of targeting and immunomasking.35 Serum halflives are also easily extended by the use of covalently attached PEG chains.36 The surface charge of the VLPs is also very important for its pharmacokinetics, and techniques have been developed to control the zeta potential easily.36,37 As well as the above, the great advantage of the MS2 SV is its pH-mediated assembly/disassembly mechanism. This allows facile self-assembly in production of the SV as well as providing a mechanism for cargoes to escape the SV in the late endosome. A number of pH-sensitive synthetic polymer formulations have been produced to mimic this behavior, but the MS2 system appears ideally set up for high cargo carrying capacity, easy assembly, and conformationally sensitive cargo release. A number of other groups are now beginning to exploit these

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*University of Leeds, Astbury Centre for Structural Molecular Biology, Leeds, LS2 9JT, U.K. Tel: 0113-343-3092. Fax: 0113343-7897. E-mail: [email protected]. Present Address †

Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, U.K. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Gareth Howell for his help with the cell imaging and Ms. Amy Barker for RNA oligo syntheses. F.A.G. is grateful to the University of Leeds and the UK BBSRC for a postgraduate studentship.

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REFERENCES

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