Multicomponent Supramolecular Polymers as a Modular Platform for

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Multicomponent Supramolecular Polymers as a Modular Platform for Intracellular Delivery Maarten H Bakker, Cameron C. Lee, E. W. Meijer, Patricia Y. W. Dankers, and Lorenzo Albertazzi ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b05383 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on January 27, 2016

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Multicomponent Supramolecular Polymers as a Modular Platform for Intracellular Delivery Maarten H. Bakker1,2, Cameron C. Lee4, E.W. Meijer1,2,3, Patricia Y.W. Dankers1,2* & Lorenzo Albertazzi1,2,5* 1

Institute for Complex Molecular Systems, Eindhoven University of Technology, 5612 AZ

Eindhoven, The Netherlands. 2Department of Biomedical Engineering, Eindhoven University of Technology, 5612 AZ Eindhoven, The Netherlands. 3Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, 5612 AZ Eindhoven, The Netherlands. 4Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139, USA. 5

Institute for Bioengineering of Catalonia (IBEC), C\ Baldiri Reixac 15-21, 08028 Barcelona,

Spain.

Corresponding Author *Corresponding author E-mail: [email protected] (P.Y.W.D), [email protected] (L.A).

ACS Paragon Plus Environment

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Abstract

Supramolecular polymers are an emerging family of nanosized structures with potential use in materials chemistry and medicine. Surprisingly, application of supramolecular polymers in the field of drug delivery has received only limited attention. Here, we explore the potential of pegylated 1,3,5-benzenetricarboxamide (BTA) supramolecular polymers for intracellular delivery. Exploiting the unique modular approach of supramolecular chemistry, we can co-assemble neutral and cationic BTAs and control the overall properties of the polymer by simple monomer mixing. Moreover, this platform offers a versatile approach towards functionalization. The core can be efficiently loaded with a hydrophobic guest molecule while the exterior can be electrostatically complexed with siRNA. It is demonstrated that both compounds can be delivered in living cells, and that they can be combined to enable a dual delivery strategy. These results show the advantages of employing a modular system and pave the way for application of supramolecular polymers in intracellular delivery.

TOC

Keywords: supramolecular; self-assembly; intracellular delivery; siRNA; hydrophobic guest; BTA; multicomponent.


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Selective delivery of therapeutic compounds to diseased target sites is an important challenge in pharmaceutical chemistry. Many of the available drugs are hindered in their therapeutic potential by critical issues such as rapid clearance by the kidneys and the reticulo-endothelial system, undesirable pharmacokinetic properties, lack of selectivity and poor cellular internalization.1–5 Nanotechnology has the potential to alter the landscape of medicine by providing targeted solutions for the delivery of both small molecule drugs and biopharmaceuticals such as proteins and nucleic acids.6–9 To achieve this, a wide collection of drug carriers has been proposed with many in clinical trials and several formulations, such as DOXIL® (doxorubicin-containing liposomes), already approved for clinical use.10 However, the potential of this approach is far from being harvested and the search for novel materials endowed with ideal delivery properties is a very active field of research. Supramolecular polymers11,12 are a novel family of nanosized structures with high potential for application in energy, materials chemistry and medicine.13 In supramolecular polymers the monomers self-assemble in one-dimensional polymers through non-covalent interactions such as H-bonding, electrostatic and hydrophobic interactions. The supramolecular nature of these materials confers them unique properties such as modularity and dynamics. Nonconvalent synthesis allows for tuning of the final properties of the aggregates and convenient incorporation of functionalities, without repeated and challenging covalent synthesis steps.14 Several examples of applications of supramolecular materials in regenerative medicine have been presented,15 for example using peptide amphiphiles,16,17 host-guest systems18 or ureidopyrimidinone materials.19 Supramolecular polymer systems have also showed promising properties for application in the field of drug delivery.20–24 In this study we aimed to investigate the applicability of a supramolecular platform in intracellular delivery using the supramolecular 1,3,5-benzenetricarboxamide derivative (BTA).


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BTA is a versatile supramolecular building block able to self-assemble in water into onedimensional (1D) aggregates.25 In our group, much effort has been devoted to understand the molecular self-assembly mechanisms and to control properties of supramolecular BTA aggregates.26,27 In particular, we studied the dynamic behavior of BTA aggregates, i.e. the ability to continuously polymerize and depolymerize, and we showed new interesting properties that derive from its dynamic behavior such as adaptivity.28,29 Multicomponent supramolecular nanofibers have been prepared by co-assembly of various functionalized monomers, showcasing the versatility and possibilities of the modularly prepared BTA-based polymers.30 BTA polymers in water display two characteristic compartments; firstly, the hydrophobic core compartment, which exhibits the ability to encapsulate small hydrophobic molecules.31 Secondly, the functionalizable hydrophilic exterior, which, when decorated with positive charges, was demonstrated to act as a dynamic multivalent binder of ssDNA via electrostatic interactions.29 Here, we study the potential of the supramolecular BTA platform for intracellular delivery and exploit both compartments of the aggregate to design a dual drug delivery system (Fig. 1a). Nile red (NR) is used as hydrophobic guest for encapsulation in the interior; NR is a solvatochromic dye that fluoresces in hydrophobic environments and is widely used as a hydrophobic model compound in drug delivery research.32–36 For electrostatic binding on the exterior, we selected short interfering RNA (siRNA). siRNA is a class of short double-stranded RNA molecules and of great therapeutic interest because it has proven to selectively regulate gene expression via the RNA interference pathway.37 However, siRNAs are susceptible to degradation and are poorly membrane permeable; carrier vehicles that protect the fragile molecules and help them breech cellular membranes to reach the cytosol are important biological and medical tools.38 Exploiting the modular approach of the BTA platform we are able to prepare functional polymers and control the overall properties by simple monomer mixing. We show the ability to load NR and siRNA on a modularly prepared


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cationic multicomponent BTA polymer and the delivery of this cargo into living cells. Furthermore, we demonstrate that siRNA delivered with the aid of cationic BTA polymers significantly reduces the expression of a targeted gene. These results prove the viability of supramolecular BTA polymers as an intracellular delivery platform and demonstrate the convenience of employing a modular system for this purpose.

Figure 1. BTA Supramolecular polymers as a platform for intracellular delivery. (a) The BTA fiber contains two compartments that can be exploited for intracellular delivery. Small hydrophobic compounds such as Nile Red can be encapsulated in the lipophilic core and siRNA can be condensed on the functionalizable hydrophilic exterior via electrostatic interactions. (b) Multicomponent polymers were prepared via co-assembly of non-functional, positively charged and fluorescently labeled BTA monomers.


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Results & Discussion Supramolecular multicomponent polymer assembly. Our self-assembling unit consists of a pegylated 1,3,5-benzenetricarboxamide (BTA) unit which directs self-assembly in water into triple hydrogen-bonded helices. A small library of BTA monomers was prepared, varying in functionalization of the PEG tails, which were used to formulate multicomponent BTA aggregates (Fig. 1b). These monomers comprise: 1) an inert BTA monomer with alcohol groups serving as scaffold for functionalized monomers to polymerize with (BTA), 2) a cationic BTA functionalized with one amine (BTA+), 3) a three-fold cationic BTA functionalized with three amines (BTA3+) and 4) a Cy5-labeled monomer as a reporter for fluorescence imaging (BTA-Cy5). The synthesis and molecule characterization of the monomers is reported elsewhere.28,29,31 Preparation of multicomponent supramolecular polymers was achieved via a simple mix and equilibration procedure. All synthesized monomers were separately dissolved in DMSO at 10 mM concentrations. These solutions of molecularly dissolved monomers were mixed in the desired ratio: the relative amount of the monomers in the mixture determines the stoichiometry of the functionalities in the final assembly. To trigger self-assembly the DMSO mixture was injected in water, typically to reach a final concentration of 100 µM and a DMSO content below 1%. Supramolecular polymers were equilibrated at room temperature for 24 hour before use. This versatile approach allows the easy and fast preparation of a library of polymers varying in physicochemical properties, functionality and multivalency, and to identify the ideal monomer composition for the desired application. Here we used this modular approach to prepare a new set of supramolecular polymers, designed for cellular uptake and intracellular delivery. Multicomponent polymers were prepared starting


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with the inert monomer as a scaffold and co-assembling various ratios (0, 25, 35, 50%) of BTA3+ monomers to promote cellular internalization. Additionally, 1% of the BTA-Cy5 monomer was coassembled in all polymers. After equilibration, dynamic light scattering and zeta potential measurements were performed (see Supplementary Table 1). The aggregates displayed hydrodynamic diameters between approximately 100-200 nm and were highly cationic when BTA3+ monomers were incorporated. The values do show the clear cationic nature of BTA3+containing aggregates and almost no effect of exact fiber composition on the hydrodynamic diameter. Notably a small error in the zeta potential distributions can be observed due to the anisotropic nature of our sample. The presence of only one zeta-potential distribution indicates that the monomers have indeed co-assembled into a multicomponent polymer, indicating that by simple mixing of different monomers the final properties of the assembly can be controlled.

Cellular interactions and cytotoxicity of BTA polymers. To investigate cellular internalization, Human Kidney (HK-2) cells were incubated with medium containing the prepared BTA polymers. Due to the co-assembly of the fluorescent BTA-Cy5, the location of the BTA fibers can be followed with confocal microscopy (Fig. 2a). After incubation and subsequent washing, no membrane binding or internalization was observed for a neutral polymer. In contrast, cationic BTA polymers initiated rapid membrane binding, with the 50% BTA3+ polymer showing the most pronounced effect. Mixing of different monomers allowed control over the properties of the polymers, which consecutively results in control over the bioactivity. Owing to the modularity of our approach, these interactions can simply be tuned. Here, in accordance with literature, charge appears to be a decisive factor and is responsible for binding via electrostatic interactions with negatively charged proteoglycans at the cell surface.39 The absence of aspecific cell binding for the non-functionalized polymer is a convenient aspect. This allows for selected interactions other than


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via non-specific charge-mediated binding, e.g. using cell-binding peptides or aptamers. Moreover the lack of BTA uptake in the case of the neutral polymer rules out the endocytosis of labeled monomers as a pathway for cellular internalization. Subsequent to membrane binding, cellular uptake was observed after approximately two hours (see Supplementary Fig. 1). When the internalization experiment was performed at 4 °C, similar membrane binding but a significant decrease in internalization was observed (see Supplementary Fig. 1). This suggests that internalization of the polymers occurs mainly via an energy-dependent endocytic process.

Figure 2. Membrane binding and cytotoxicity of BTA polymers. (a) Internalization of 10 µM (cationic) BTA polymers. Co-assembly of a percentage of BTA3+ monomers with inert monomers results in binding to the cell membrane in 30 minutes and subsequent internalization of the otherwise cell-impermeable BTA aggregates. Nuclei are stained with Hoechst. BTA polymers are visualized in white, originating from the co-assembled BTA-Cy5 monomer. Scale bar represents 25 µm. (b) Viability of Human Kidney cells after 24 hour incubation with BTA polymers coassembled with increasing percentages of the BTA3+ monomer and for a homopolymer consisting


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entirely of the BTA+ monomer. A range of concentrations was tested. Highly cationic polymers induce a decrease in cell viability that correlates with charge density and concentration. Values indicate mean + SD, n=5. It is well known that polycationic drug carriers cause cell damage and cell death through processes like plasma membrane damage, mitochondrial permeabilization, late phase lysosomal perforation and alkalinization.40 We investigated whether internalization of our BTA polymers causes cytotoxicity and if this should be attributed to the BTA core or to the positive charges. Cell viability was assessed with an MTT assay on HK-2 cells incubated with BTA polymers. We compared polymers varying the fraction of BTA3+ monomers co-assembled (0, 10, 25, 35 and 50%) for a range of concentrations (Fig. 2b). As expected, neutral BTA polymers had no influence on cell viability for all concentrations studied. Moreover, no toxicity was observed for a 10% and 25% BTA3+ polymer over the whole range of concentrations tested. This suggests that the BTA moiety by itself exhibits no cytotoxic effect. On the other hand, highly cationic materials induced a significant reduction in cell viability that correlates with charge density and concentration, similar to the known cytotoxic effects of covalent cationic materials.41 Notably, the difference in cytotoxicity between polymers is not merely related to total charge, but is strongly modulated by charge density along the fiber. A 25% BTA3+ polymer at a 20 µM concentration introduces the same total amount of amines as a 50% BTA3+ polymer at 10 µM, but has a lower charge density and is significantly less toxic. To further investigate this, cytotoxicity of a homopolymer of the BTA+ was evaluated. This polymer has its charges more evenly spread but effectively exhibits an identical overall charge density as the 35% BTA3+ polymer. We observed no significant difference in toxicity between these polymers, confirming that indeed the overall charge density along the fiber is the crucial parameter for toxicity.


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In essence, the density of cationic charge controls both cell binding and toxicity and an ideal balance between these two has to be found. In this framework the modularity of the supramolecular approach allows an easy composition screening for fine tuning of cell binding and toxicity. In this study, the remaining cell experiments with cationic polymers were performed with concentrations and incubation times that lie within the non-toxic range.

Intracellular trafficking of BTA polymers. In order to investigate the trafficking of the polymers upon internalization, time-lapse imaging was performed on HK-2 cells incubated with 50% BTA3+ fibers co-assembled with 1% BTA-Cy5 (Fig. 3). Subsequently to the initial rapid membrane binding, two main stages in the intracellular trafficking were identified. Firstly, after approximately two hours, most of the polymers were transferred from the membrane to the perinuclear region, which resembled the distinct shape of the endoplasmic reticulum (ER). In the second phase, after approximately twelve hours of incubation, the BTAs were mainly concentrated in vesicular structures, which did not appear to change any further over time. To identify the exact compartments involved in these stages of internalization, colocalization studies with organelle markers were performed. Three markers were imaged at all time points and the ones exhibiting strong colocalization are shown (Fig. 3). Firstly, using a plasma membrane staining the initial membrane binding was confirmed. To verify the location of the polymers after two hours, we used an ER-Tracker and indeed observed partial colocalization. In the final stage, we determined the nature of the vesicles to be lysosomes by the strong colocalization with LysoTtracker. Commonly, macromolecules that enter cells via endocytosis are eventually trafficked to lysosomes, similar as observed here for the BTA molecules. However, transportation in between generally occurs via early endosomes that transfer to late endosomes and finally mature into


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lysosomes.42 Lately, more attention is dedicated towards endocytic internalization and intracellular trafficking pathways, as there are strong indications that an inability to escape the endolysosomal system is a key limiting factor for cellular uptake and drug delivery carriers.43–45 While this stream of intracellular trafficking to the lysosomes is not exclusive, a degree of colocalization with the ER that is shown here is uncommon. More intensive research into the trafficking and localization of BTAs is necessary to clarify the exact pathway. Also, it is currently unclear whether the BTA polymers remain in an aggregated state or are depolymerized after cellular binding and internalization. Confocal microscopy cannot identify this due to the lack of resolving power.

Figure 3. Intracellular trafficking of BTA polymers. Time-lapse imaging of HK-2 cells incubated with 10 µM 50% BTA3+ aggregates, labeled with 1% BTA-Cy5. Colocalization studies are


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performed and demonstrate rapid membrane binding within the first hour. After two hours incubation the supramolecular material is present throughout the cell and shows colocalization with the ER. Hours later, the BTAs have been transferred to lysosomes, as is observed from the strong colocalization with a lysosome marker. Nuclei are stained with Hoechst. Scale bars represent 10 µm.

Loading and intracellular delivery of a hydrophobic guest and siRNA. Cationic BTA polymers proved to be able to cross the cellular membranes via charge-mediated endocytosis, the first step towards intracellular delivery. In order to deliver a therapeutic molecule, the polymer should be able to be loaded with a cargo without compromising its internalization properties. Since siRNA is of polyanionic nature, condensation of siRNA can be achieved via electrostatic complexation with primary amines present in cationic carriers and is described by the N/P ratio (ratio of moles of amine in carrier to moles of phosphates in siRNA). In order to investigate the ability of a cationic BTA polymer to condense siRNA, an ethidium bromide (EtBr) displacement assay and a gel retardation assay were performed. Upon electrostatic complexation of siRNA with the charged BTA polymer, EtBr was displaced from the intercalation cavities between RNA base pairs and the associated loss of fluorescence emission correlated with the N/P ratio (Fig. 4a). Similar observations were made with a gel retardation assay; where complete retardation of siRNA was reached with N/P ratio ≥ 6 (see Supplementary Fig. 2). Altogether, these results indicate that an N/P ratio of at least six is sufficient to condensate siRNA into stable complexes. Next, the ability of BTA polymers to encapsulate small organic molecules in the hydrophobic core of the fiber was probed using nile red (NR). Titration of NR to a 25 µM 50% BTA3+ polymer in aqueous solution gives a maximum encapsulation efficiency of 4 µM NR (Fig. 4b). This value


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translates to a NR loading of almost 4% w/w, which is amongst the higher values reported in literature.34–36 Additional DLS and zeta measurements were performed to study whether siRNA complexation or NR encapsulation influences the overall structure or properties of the BTA fiber. The 50% BTA3+ fiber was measured in complex with siRNA (N/P ratio 10), in complex with NR, and in complex with both NR and siRNA. All complexes exhibited values for hydrodynamic diameter and zeta potential in the same region as the original fiber (see Supplementary Fig. 3). Furthermore, cryoTEM on the 50% BTA3+ polymer in complex with siRNA still showed the characteristic fibrillar aggregates (see Supplementary Fig. 4), similar as previously reported BTA fibers.31

Figure 4. Loading and intracellular delivery of siRNA and NR with BTA polymers. (a) An EtBr displacement assay shows that siRNA can be effectively condensed. (b) NR is used as a model hydrophobic compound and shows a loading of up to 4% w/w. (c) Directly added (37.5 nM) free


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fluorescent siRNA is not internalized by HK-2 cells. (d) On the other hand, when 10 µM 50% BTA3+ is used as a transfection agent a large amount of the fluorescent siRNA is internalized. (e) Addition of free NR (0.5 µM) to HK-2 cells. (f) Cells incubated with NR formulated in the core of a 10 µM 50% BTA3+ polymer, a significant increase in intracellular delivered NR is observed compared to free addition. Scale bars represent 100 µm. Having demonstrated the capability to load either siRNA or NR as cargo, we then tested the ability to deliver these molecules into living cells. Addition of naked fluorescently labeled (Alexa488) siRNA to HK-2 cells shows negligible internalization due to the unfavorable interactions with the negatively charged cell membranes (Fig. 4c). In contrast, when siRNA is added in an electrostatic complex with cationic BTA polymers, siRNA is delivered to the interior of the cells (Fig. 4d). Similarly, addition of free NR to HK-2 cells results in poor internalization (Fig. 4e). But, when incubated after encapsulation in BTA polymers, a sharp increase of NR inside the cells is observed (Fig. 4f). Importantly, because both compounds are loaded in different compartments of the BTA polymer, they can also be loaded and delivered simultaneously (see Supplementary Fig. 5). In order to understand the mechanism of delivery 2-color confocal imaging was performed, tracking simultaneously the carrier and the payloads. Interestingly, NR dissociated from the polymers and crossed the membrane almost instantly at the moment of membrane binding, indicating that BTA polymers merely serve as a solubilizer for NR until it can transfer to more hydrophobic intracellular lipid compartments (see Supplementary Fig. 6). On the contrary, siRNA and BTA can still be seen partly colocalized, both on the membrane and once internalized (see Supplementary Fig. 6).


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Inducing gene knockdown with a functional siRNA. We have demonstrated the efficient loading and intracellular delivery of the BTA carrier and its cargo using a model hydrophobic compound and a fluorescent siRNA. To demonstrate the relevance of this approach the fluorescent siRNA was replaced with a functional siRNA and gene silencing efficacy was investigated. RNA silencing experiments were performed with siRNA targeting the ELAV1 encoding mRNA using a selection of cationic BTA polymers as transfection agent. Naked added siRNA serves as a negative control and showed no decrease in mRNA expression (Fig. 5). On the other hand, all transfections performed using cationic BTA polymers resulted in a significant reduction in ELAV1 mRNA levels after 48 hour. In particular the 50% BTA3+ and the 100% BTA+ transfections showed a pronounced decrease of mRNA expression. The 100% BTA+ polymer displays a higher silencing efficacy compared to the 35% BTA3+ polymer; it is hypothesized that this can be attributed to a more effective complex formation due to the better spreading of charges on the BTA+ polymer.

Figure 5. Gene knockdown with BTA-mediated siRNA delivery. ELAV1 mRNA expression of HK-2 cells 48 hour after siRNA transfection with cationic BTA polymers, normalized to GAPDH mRNA expression. Transfections were performed with 35 pmol siRNA complexed at N/P ratio =


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10. This translates to final BTA concentrations of 18.6, 13.3, 9.3 and 14.0 µM for 25% BTA3+, 35% BTA3+, 50% BTA3+ and 100% BTA+ polymers, respectively. Lipo = Lipofectamine-2000. All samples led to a decrease in gene expression compared to naked added siRNA. Values indicate mean + SD.

The maximum of 41% silencing efficacy with cationic BTAs is lower than the 68% silencing that was reached using lipofectamine-2000. Lipofectamine is a golden standard in in vitro siRNA transfection and was included here as positive control. Generally, silencing efficacies with lipofectamine are expected to be higher, implying that these cells or the targeted gene in these cells are difficult to silence. Nonetheless, the decreased mRNA levels for all BTA transfections demonstrate that the intracellular delivered siRNA has been processed and RNA interference was successfully induced. Further optimization of this system is needed to fine tune the balance between charge and charge density with toxicity and transfection efficacy. Work by Zhou and colleagues serves as an example of ways to manipulate this balance.46 Zhou reduced charge density of their polymers -and thereby toxicity- and managed to maintain function by increasing molecular weight and hydrophobicity. Here, possible improvements can conveniently be implemented owing to the modularity of the system, which allows easy introduction of e.g. targeting moieties, more effective siRNA binders, endosomal escape agents or other functionalities.

Conclusion In summary, we have introduced the supramolecular BTA platform as a convenient and viable strategy for intracellular delivery. Multicomponent supramolecular polymers were prepared by a simple mixing approach that displayed specific binding and fast cellular uptake when cationic


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monomers were co-assembled. Electrostatic condensation of siRNA and encapsulation of a small hydrophobic molecule in two different compartments of the BTA was demonstrated, which allows for a dual delivery strategy. Both siRNA and the hydrophobic molecule were successfully delivered to living cells. The BTA polymers were shown to be not inherently cytotoxic unless a high amount of cationic charges with a high density was present. Optimization of the BTA assemblies would be beneficial to fine-tune charge and charge density in respect to cytotoxicity versus bioactivity. Owing to the modularity of the supramolecular BTA system, possible adjustments can conveniently be implemented. Finally, silencing experiments showed that intracellular delivered siRNA significantly reduced gene expression, thereby confirming the potential of the supramolecular BTA platform for intracellular delivery and paving the way for further research into this topic.

Materials & Methods General. All reagents and chemicals were obtained from commercial sources at the highest purity available and used without further purification unless stated otherwise. BTA monomers were synthesized via previously reported synthesis routes.28,29,31 Alexa-488 labeled Allstars negative siRNA was purchased from Qiagen. ER-Tracker Green, LysoTracker Red DND-99 and CellMask Deep Red Plasma Membrane Stain were purchased from LifeTechnologies. High Pure RNA Isolation Kit was purchased from Roche Life Science. iScript cDNA synthesis kit and IQ SYBR Green Supermix were purchased from Bio-Rad. ELAV1 siRNA was provided by Novartis, the sequence

is

as

follows-

antisense:

UuAAUuAUCuAUUCCGuACuu,

sense:

GuAcGGAAuAGAuAAuuAAuu, where upper case denotes an RNA nucleotide and lower case denotes a 2’-OMe modified nucleotide. ELAV1 qPCR primers were purchased from Eurofins


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Genomics (fw: 5’-TAAGGTGTCGTATGCTCGCC-3’, rev: 5’-CGTCCTTCTGGGTCATGGTC3’). Water was purified on an EMD Millipore Milli-Q Integral Water Purification System. Fluorescence data were recorded on a Varian Cary Eclipse fluorescence spectrometer using Quartz cuvettes. Fluorescent cell images were acquired with a Leica TCS SP5 AOBS equipped with a 40x water immersion objective and a temperature-controlled incubation chamber maintained at 37°C. DLS data was acquired on a Zetasizer µV. Zeta-potential data was acquired on a Zetasizer Nano ZS. Reverse transcription was carried out with a BioRAD MJ Mini Thermal Cycler. qPCR was performed on a Bio-Rad CFX96 Real-Time PCR Detection System. Absorbance of MTT was read out on a Tecan Saffire 2 plate reader. Agarose gels were imaged using a GE ImageQuant 350. Formulation of BTA Aggregates. Synthesized BTA monomers were dissolved in DMSO at a concentration of 10 mM. For preparation of the aggregates the desired amounts of monomer solutions were pre-mixed in a vial to obtain a mixture of monomers that are still in a molecularly dissolved state. To trigger self-assembly the DMSO mixture was injected in water with 5 mM HEPES buffer, typically to reach a final concentration of 100 µM and a DMSO content below 1%. Samples were equilibrated at room temperature for 24 hours before experiments. For complex formation with siRNA the appropriate amount of siRNA from a 20 µM stock solution in Milli-Q was injected into a pre-assembled BTA polymer solution. The sample was then equilibrated for 3 more hours by means of shaking at room temperature. Cell Culturing & Imaging. Human Kidney epithelial (HK-2) cells were purchased from ATCC and cultured at 37° C in 95% air/5% CO2 atmosphere in Dulbecco’s Modified Eagle Medium (DMEM) 41965-039 supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin (Pen/Strep). Cells were passed typically twice a week and for experiments cells ranging from passage 5 to 25 were used. For fluorescence imaging cells were trypsinized and


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seeded in 8-well Thermo Scientific Lab-Tek Chambered Coverglass (10.000 cells/well) 24 hour in advance. Upon imaging, the media was removed and replaced by 10 µM BTAs dissolved in DMEM supplemented with 2% FBS. After the reported incubation times, the media was removed and cells were washed twice with PBS and incubated with fresh media supplemented with 10% FBS and 1% Pen/Strep. In colocalization studies the organelle markers were used following manufacturers’ protocol. Images were analyzed using ImageJ. Probing Complex Formation. For the ethidium bromide displacement assay fixed amounts of siRNA (2 µg/mL) and EtBr (3 mg/mL) were mixed in Milli-Q water. Samples were prepared containing the amounts of 50% BTA3+ polymer to obtain the desired N/P ratios (0, 0.5, 1, 2, 5, 10, 15). EtBr was excited at 510 nm and its fluorescence recorded at 595 nm. Fluorescence intensity was normalized to the sample with N/P = 0. For the gel retardation assay samples with increasing N/P ratios (0, 0.25, 0.5, 1, 2, 4, 6, 10) were prepared with 50 ng siRNA and pre-assembled 50% BTA3+ polymer solutions in a total volume of 25 µl. Samples were then run on a 1.5 wt% agarose gel containing EtBr on 90 volt for 45 minutes and subsequently on 20 volt for 15 minutes. Gels were imaged with the EtBr filter on an ImageQuant 350. Nile Red Titration. Measurements were performed with a 25 µM 50% BTA3+ polymer solution. NR titration was performed with concentrations between 0 and 8 µM. Each time upon increasing the NR concentration the sample was equilibrated for 10 minutes. NR was excited at 550 nm and the fluorescence recorded at 590 nm. Maximum emission intensity values are plotted versus NR concentration. MTT assay. HK-2 cells were seeded in 96 well plates (5000 cells/well) and cultured in DMEM media supplemented with 10% FBS and 1% Pen/Strep overnight. The next day the media was removed and 166 µl of BTA polymers dissolved in DMEM media supplemented with 2% FBS was added and incubated at 37 °C. Each condition was performed in 5-fold. After approximately 24


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hour incubation, 18 µL of freshly prepared MTT solution (5 mg/mL in PBS, filtered through 0.2 µm filter) was added to the wells and incubated for 2 hours. The medium was then removed and replaced with 0.04 M HCl in isopropanol and incubated for 1 hour. After gently mixing, 100 µl of the isopropanol solution was transferred to a Costar EIA/RIA 96 wells plate. The absorbance was measured at 570 nm and the absorbance at 650 nm was deducted from this value. Values were normalized to non-BTA treated cells and reported as average and standard deviation. Transfection and evaluating knockdown. HK-2 cells were seeded in a 24 wells plate (30000 cells/well) and cultured in DMEM media supplemented with 10% FBS and 1% Pen/Strep overnight. The next day BTA-siRNA complexes were mixed with media supplemented with 2% FBS to a final volume of 1 ml. All samples contained 35 pmol siRNA and were complexed with the various BTA polymers at N/P ratio = 10. Cells were washed with PBS once and subsequently treated with the 1 ml BTA-siRNA samples. After 4 hours incubation, media containing BTAsiRNA complexes was discarded and replaced by fresh DMEM supplemented with 10% FBS and 1% Pen/Strep. Lipofectamine-2000 transfection was performed according to manufacturers’ protocol. Approximately 48 hours after transfection, RNA of the cells was extracted using a High Pure RNA Isolation Kit following manufacturers’ protocol and immediately afterwards transcribed to cDNA with an iScript cDNA synthesis kit following manufacturers’ protocol. Quantitative polymerase chain reaction was performed with 5 ng cDNA and ELAV1 specific primers to quantify ELAV1 mRNA expression. Normalization of each sample was performed to the expression of the housekeeping gene GAPDH. Reported expression values of BTA and Lipofectamine-2000 transfections are averages and standard deviation from a total of 11-12 samples per condition, based on 3 independent experiments, normalized to ELAV1 expression of untreated cells. Reported expression of cells treated with naked siRNA is the average and standard deviation of 4 samples, normalized to ELAV1 expression of untreated cells.


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CryoEM. Vitrified films were prepared in a ‘Vitrobot’ instrument (PC controlled vitrification robot, patent applied, Frederik et al 2002, patent licensed to FEI) at 22°C and at a humidity of 100%. In the preparation chamber of the ‘Vitrobot’, a 3µl sample was applied on a Quantifoil grid (R 2/2, Quantifoil Micro Tools GmbH), which was surface plasma treated just prior to use (Cressington 208 carbon coater operating at 5 mA for 40 s). Excess sample was removed by blotting using filter paper for 3 s at –3 mm, and the thin film thus formed was plunged (acceleration about 3 g) into liquid ethane just above its freezing point. The vitrified film was transferred to a cryoholder (Gatan 626) and observed at temperatures below -170 °C in a Tecnai Sphera microscope operating at 200 kV. Micrographs were taken at low dose conditions, using a defocus setting of 5 µm at 25000 magnification

Supporting Information. DLS, zeta potential measurements, temperature-dependent internalization images, agarose gel retardation assay, Cryo-TEM and more nile red and siRNA delivery images are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgements. The research leading to this manuscript has received funding from the Ministry of Education, Culture and Science (Gravity program 024.001.035), the Netherlands Organization for Scientific


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Research (NWO), the European Research Council (FP7/2007-2013) ERC Grant Agreement 308045, and the Netherlands Institute for Regenerative Medicine (NIRM). L.A is grateful for financial support from the Netherlands Organization for Scientific Research (NWO – VENI Grant: 722.014.010). The authors thank R.P.M. Lafleur for Cryo-TEM measurements.

Competing financial interests. The authors declare no competing financial interests. References (1) (2) (3) (4) (5) (6) (7)

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