Hierarchical Assembly of siRNA with Tetraamino Fullerene in

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Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Hierarchical Assembly of siRNA with Tetraamino Fullerene in Physiological Conditions for Efficient Internalization into Cells and Knockdown Kosuke Minami,†,# Koji Okamoto,‡ Koji Harano,*,† Eisei Noiri,*,‡ and Eiichi Nakamura*,† †

Department of Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Department of Nephrology and Endocrinology, University Hospital, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

‡

S Supporting Information *

ABSTRACT: Delivery of siRNA is a key technique in alternative gene therapy, where the siRNA cargo must be effectively loaded onto a tailordesigned carrier molecule and smoothly unloaded precisely upon arrival at the target cells or organs. Any toxicity issues also need to be mitigated by suitable choice of the carrier molecule. A water-soluble cationic fullerene, tetra(piperazino)[60]fullerene epoxide (TPFE), was previously shown to be nontoxic and effective for lung-targeted in vivo siRNA delivery by way of agglutination-induced accumulation. We found in this in vitro study that hierarchical reversible assembly of micrometer-sized TPFE−siRNA−serum protein ternary complexes is the key element for effective loading and release, and stabilization of otherwise highly unstable siRNA under the physiological conditions. The amphiphilic TPFE molecule forms a sub-10 nm-sized stable micelle because of strong cohesion between fullerene molecules, and this fullerene aggregate protects siRNA and induces the hierarchical assembly. Unlike popularly used polyamine carriers, TPFE is not toxic at the dose used for the siRNA delivery. KEYWORDS: hierarchical assembly, gene delivery, siRNA, cell internalization, nanomedicine, fullerene

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INTRODUCTION RNA interference (RNAi)1 is a post-transcriptional sequencespecific gene-silencing phenomenon of great therapeutic potential.2 Small interfering RNAs (siRNAs) are small fragments of RNA (21−23 nucleotide length) used for RNAi. They are, however, unstable under the physiological environment and show low membrane permeability because of their negative charges and hydrophilicity. To utilize siRNA for therapeutic applications, the siRNA cargo must be effectively loaded onto a nontoxic carrier molecule and smoothly unloaded precisely upon arrival at the target cells or organs.3 Liposomes made of cationic lipids have been widely used to enhance interactions with negatively charged oligonucleotides, including siRNA,4 while they often show dose-dependent toxicity. Discovery of the interaction between a water-soluble [60]fullerene derivative with a double-stranded DNA5 in 1993 stimulated quick development of nanomedicinal research using fullerenes,6−8 culminating recently in in vivo delivery of DNA,9 and siRNA targeted specifically to the lung using a water-soluble cationic fullerene, tetra(piperazino)[60]fullerene epoxide (TPFE).10 TPFE showed hierarchical self-assembly to form a nanoscale ternary TPFE−siRNA−plasma protein complex (Figure 1). This ternary complex structure shows not only rapid accumulation in lung capillary but also fast © XXXX American Chemical Society

clearance within 24 h from the lung without showing any pulmonary toxicity. The clearance occurs through the disintegration of the ternary complex, which releases siRNA into the cell interior. Ascertaining the reason for such efficiency will provide a basic understanding of the mechanism of organized assembly of RNA and its application to nanomedicine. Here, we report the results of chemical, microscopical, and in vitro biological study on the ternary complex to show that the observed efficiency of the loading and unloading of siRNA originated from the ability of TPFE to form a stable sub-10 nm-sized micelle,11,12 on which siRNA molecules are hierarchically assembled. This assembly contributed to the stabilization of siRNA against enzymatic degradation, and also to the high efficiency of siRNA delivery into cells with low cytotoxicity. The sizes of this aggregate can be tuned by changing the ratio (R = fullerene molecule/base pair) between 21-mer double-stranded siRNA and TPFE (bearing four basic nitrogen atoms), and the optimum value of R in vitro was found to be the same as that for in vivo delivery. The nanoscale ternary TPFE−siRNA−plasma protein complex was found to provide not only efficient protection from enzymatic Received: January 31, 2018 Accepted: May 9, 2018 Published: May 9, 2018 A

DOI: 10.1021/acsami.8b01869 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of ternary complex formation from unit structure of self-assembled TPFE.

analysis. One was sub-10 nm aggregates at 9.1 nm and the other sub-micrometer-sized aggregates centered at 310 nm (Figure 2b, red line). The sub-10 nm aggregates can be separated by centrifugation as a fraction of unimodal distribution with an average diameter of 7.2 nm, which did not show any size change for 12 h (Figure 2b, blue line), indicating that the sub10 nm aggregates are kinetically stable under the acidic environment. The aggregated structure of TPFE was analyzed by STEM without recourse to staining because [60]fullerene by itself produces a strong contrast. The STEM image showed small aggregates as black dots, and large gray objects in rounded shapes (Figure 2c). The dots were 5.4 ± 2.9 nm in diameter (Figure 2c; N = 439), and showed high contrast, indicating that the TPFE molecules are densely packed. On the other hand, the objects larger than 100 nm with low image contrast can be considered to correspond to the large aggregates seen in the DLS and LD Analysis. The lower contrast of the large aggregates suggests that TPFE molecules in the larger aggregates are loosely associated in the aqueous medium, and the aggregates shrunk upon drying on the carbon film. We previously showed that conical pentasubstituted fullerenes bearing ionic groups12 saccharide moieties in water28 form stable micelles of 4−8 nm in diameter because of the strong cohesive force between the fullerene molecules. In addition, pristine [60]fullerene forms a close-packed crystalline structure with a small number of C60 molecules (C60)n because of the unique spherical structure.29 Therefore, we consider that the TPFE molecules also assemble to form a micelle 7 nm in diameter (Figure 2a). Binding Efficiency of TPFE with siRNA. We previously reported10 that the TPFE−siRNA complex with a TPFE/ siRNA ratio (R) of 20 (i.e., 20 molecules/base pair and 80 basic nitrogen atoms/base pair) shows significantly high knockdown efficiency and fast clearance in vivo. We were intrigued by the relationship between the structure of the TPFE/siRNA complex and the R value, and examined the loading efficiency of TPFE and the TPFE−siRNA complexes under different R values. The binding efficiency of siRNA was determined by electrophoresis. Enhanced green fluorescent protein (EGFP)targeting siRNA (siGFP; 21-mer double stranded RNA of the sequence shown in Table S1 of the Supporting Information) was dissolved in water, and the resulting aqueous solution of siGFP was mixed with an acid-buffered solution of TPFE with

degradation of siRNA but also a mechanism of fast clearance in vivo after the release of siRNA in the cytoplasm by kidney clearance.

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RESULTS AND DISCUSSION Self-assembly of TPFE in Aqueous Media. Fullerene is unique for its spherical structure, fused π-surface, and high hydrophobicity. Because of these properties, fullerenes exhibit a large variety of self-assembled behaviors, such as formation of particles,13,14 fibers,14,15 micelles,11,12 and vesicles.16−19 They thus produce nanostructures together with drug molecules20 and oligonucleotides21−23 that are useful for drug delivery.24−27 We first investigated the aggregation behavior of TPFE under aqueous environment by dynamic light scattering (DLS) and scanning transmission electron microscopy (STEM). The four piperazine groups in TPFE are protonated under acidic or pH 7 conditions. Therefore, these groups gathered together on the one side of the molecule to make a TPFE amphiphilic aggregate in water (Figure 2).9,10,22,23 TPFE dissolved in an acidic buffer (pH 2.0; KCl, 2.0 mM) showed two DLS peaks by CONTIN

Figure 2. Self-assembled aggregates of TPFE in water. (a) DLS analysis of free TPFE aggregates in acidic buffer. Red and blue lines indicate the TPFE aggregates in as-prepared solution and in the supernatant after centrifugation, respectively. (b) STEM image of free TPFE aggregates on a thin carbon film. Scale bar is 100 nm. White arrows indicate the sub-10 nm aggregates of TPFE. (c) Histogram of the sub-10 nm TPFE aggregates determined by STEM. The average size is 5.4 ± 2.9 nm (N = 439). B

DOI: 10.1021/acsami.8b01869 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces an R value ranging between 1 and 50 (see Experimental Section). Free TPFE and TPFE−siRNA complexes were observed as a brownish-yellow colored band at sample-loading wells in the bright-field images (Figure 3a,b) because of the

STEM indicate that the TPFE aggregates and siRNA are connected through electrostatic interactions in an aqueous medium maintaining the 10-nm-sized unit structure of the primary TPFE aggregate. The loosely assembled sub-micrometer-sized structure of the TPFE−siRNA complexes disintegrated into stable sub-10 nm-sized TPFE micelles with siRNA under the condition of AFM and STEM imaging on a solid substrate (Figure 3d,e). The binding efficiency of TPFE to oligonucleotides differs greatly between RNA and DNA. Similarly to the TPFE−siRNA complexes, 21-mer double-stranded DNA (dsDNA) including two-base 5′ overhangs (see Table S1 for the sequence) also forms sub-micrometer-sized aggregates with TPFE in the range of R values from 1 to 50 (Figure S1). However, the loading efficiency determined by electrophoresis was extremely low even at the high R value of 50 (up to 50%). We assume that rigid dsDNA (mainly B-form double helix)31 and TPFE associate tightly to form sub-micrometer-sized globules and suppressed further hierarchical assembly, whereas the flexible siRNA (mainly A-form double helix)31 can cover the TPFE micelle surface to form loosely assembled structures with TPFE, and hence the assembled structures were dramatically affected by the different R values (supported by DLS analyses in Figure 3c). Ternary Complex Formation of TPFE−siRNA with Serum Proteins and Stabilization of siRNA. A key factor in the in vivo lung-selective delivery of siRNA by TPFE was spontaneous self-assembly of TPFE−siRNA with serum proteins in the bloodstream.10 The sub-micrometer-size TPFE−siRNA complexes at R = 20 rapidly agglomerate with plasma proteins in the blood vessel to form micrometer-sized globules (ca. 6 μm), which are efficiently trapped in narrow lung capillaries (ca. 5 μm in diameter) and release siRNA into lung cells.10 To monitor how TPFE protects siRNA against enzymatic degradation by addition of serum proteins, we prepared in vitro the ternary complex of TPFE−siRNA−plasma proteins. An acid-buffered solution of TPFE was mixed with siRNA; subsequently, the plasma proteins were added and then the sizes of the complexes were measured by DLS and laser diffraction (LD) analyses (Figure 4a). Although the TPFE− siRNA complexes with R = 20 did not agglomerate after 6 h in the absence of serum (white bars), TPFE−siRNA complexes significantly agglutinated with plasma proteins to form micrometer-sized globules within 5 min (6.02 ± 0.38 μm) and maintained their sizes for at least 6 h. The TPFE−siRNA complexes with serum proteins were also analyzed by highresolution SEM on a cleaned indium tin oxide (ITO)/glass substrate without conductive metal coating to observe nanoscale structural details.20 SEM images show the ternary complexes of TPFE−siRNA−plasma proteins as a submicrometer-sized agglomerate of 10−30-nm-sized particles, not globules with a size on the order of micrometers (Figure 4b). These small particles are likely to be TPFE unit structures, considering their structural robustness observed in the DLS and STEM. These results indicate that the TPFE−siRNA−serum protein ternary complexes are stable in a soluble form, but they disintegrate on a solid substrate. This observation agrees with the hypothesis advanced in our previous report10 that the TPFE−siRNA complex is loosely agglutinated with serum proteins to form micrometer-sized globules, accumulates in narrow lung capillaries, and finally disintegrates into plasma proteins and TPFE−siRNA complexes possibly by mechanical factors, such as bloodstream. The facility of this final process

Figure 3. Physicochemical characterization of TPFE−siRNA complexes. (a, b) Gel electrophoresis of TPFE−siRNA complexes with the R value ranging from 0 (siRNA only) to 50 by changing the amount of TPFE (a) and siRNA (b). BF and UV indicate bright-field and UV images, respectively. Brownish-yellow colored bands indicate the color of TPFE in BF images. Naked or unbound free siRNA was stained with ethidium bromide and observed in fluorescent images (UV). (c) DLS analysis of TPFE−siRNA complexes with the R value. Error bars indicate ± SD. Red bars indicate the observation of precipitates. (d) AFM image of TPFE−siRNA complex (R = 20) on a mica substrate. The yellow dashed circle indicates the observed flower-like structure. Scale bar is 1 μm. (e) STEM image of TPFE−siRNA complex (R = 20) on the carbon film. Blue dashed circles indicate the observed flower-like structures. Scale bar is 500 nm.

brownish-yellow color of TPFE. In the same lanes, naked siRNA was observed when the R value was between 1 and 10. In contrast, the bands of unbound siRNAs were not observed at R values of 20 and 50. These results indicate that siRNA was not bound completely to TPFE with R values smaller than 10, but fully bound to TPFE at the R value over 20. The data agree with our previous observation that high loading efficiency of siRNA into TPFE−siRNA complexes leads to high knockdown efficiency in vivo.10 The TPFE−siRNA complex was also analyzed by DLS, atomic force microscopy (AFM), and STEM (Figure 3c−e). DLS analysis showed that micrometer-sized aggregates formed at R values between 2 and 10 (red bars), and then they gradually precipitated, while the complexes formed at R = 20 and 50 formed well-dispersed sub-micrometer-sized particles (blue bars)a size suitable for cellular uptake by way of enhanced permeation and retention effects.30 AFM (Figure 3d) and STEM (Figure 3e) images of the TPFE−siRNA complexes (R = 20) showed that small aggregates gathered to form a flower-like structure on a substrate with sizes of about 500 nm (dashed circles in Figure 3d,e). The height of the flower-like objects obtained in this AFM image was ca. 100 nm, suggesting that several unit-aggregated structures of TPFE assembled together with siRNA under the condition of the AFM measurement. Note that the samples of TPFE−siRNA complexes for STEM were taken again without staining, indicating that the flower-like objects are aggregates of the highcontrast TPFE molecules. These results observed by AFM and C

DOI: 10.1021/acsami.8b01869 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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time reverse-transcription polymerase chain reaction (RTPCR). The TPFE−siRNA complexes (R = 5, 10, and 20) were transfected into the EGFP-overexpressing human embryonic kidney 293 (HEK293) cell (293-GFP). An EGFP-targeted chemically stabilized siGFP (Stealth siGFP), which has resistance against enzymatic degradation, was also investigated for comparison of efficiency with nonstabilized siRNA. Lipofectamine 2000, a commercially available transfection reagent of siRNA, was also used for the transfection agent as a reference. We also tested nontargeting siRNA (siNEG) as a negative control to evaluate nonspecific gene silencing, socalled “off-target effect”.33 Among the three R values tested, the R value of 20, which showed efficient siRNA delivery in vivo,10 obtained the highest knockdown efficiency (61% for Stealth siGFP and 51% for siGFP), and was higher than that of Lipofectamine 2000 (49% for Stealth siGFP and 35% for siGFP) (Figure 5). It should be

Figure 4. Effective stabilization of siRNA by the formation of TPFE− siRNA complexes. (a) DLS and LD analyses of TPFE−siRNA complexes in the presence or absence of serum proteins (R = 20). (b) SEM image of TPFE−siRNA−serum protein ternary complexes on an ITO/glass substrate without conductive metal coatings. Scale bar is 100 nm. (c) Stabilization of siRNA by TPFE against enzymatic degradation in the simulated physiological environment analyzed by electrophoresis. TPFE and TPFE−siRNA complexes were observed in the BF images. Naked or unbound free siRNA was stained with ethidium bromide and observed in fluorescent images (UV). The condition is shown in the table below. “Serum” indicates the addition of serum protein (+), or no addition of serum protein (−). “Extraction” indicates that samples were loaded as a form of TPFE− siRNA complex (−), or were loaded after extraction of siRNA from TPFE−siRNA complexes by modified TRIzol reagents (+). See also Experimental Section. (d) Percentage of free siRNA calculated from the contrast of electrophoresis image (c). Numbers correspond to that in electrophoresis.

Figure 5. RNAi activity and cytotoxicity of TPFE in vitro. (a) Knockdown efficiency of TPFE-based siRNA delivery in vitro. Three siRNAs were used: siNEG, nontargeting siRNA (black); siGFP, EGFP-targeting siRNA (white); Stealth siGFP, EGFP-targeting chemically stabilized siRNA (gray). As a reference, commercially available transfection reagent, Lipofectamine 2000 (Lipo2000), was used. The TPFE/siRNA ratio of R was varied in the range of 5 to 20. Error bars are ± SD. **P < 0.01 vs siNEG. (b) Cytotoxicity assay of TPFE−siRNA complexes. siNEG (red) and siGFP (blue) were analyzed. Error bars are ± SD. Fitting curves were calculated by the dose-dependent logistic model.

accounts for the efficient internalization of the siRNA into lung cells. In the siRNA delivery, carrier vehicles must protect siRNAs from enzymatic degradation under the physiological environment. We evaluated the stabilization of siRNA by TPFE by analyzing the amount of remaining siRNA in TPFE−siRNA complexes under exposure to serum media (see also Experimental Section). The amount of extracted siRNA was measured by electrophoresis (Figure 4c; see also Figure S2). TPFE−siRNA complex (R = 20) was incubated for 15 min in the diluted mouse serum as a simulated physiological environment.32 The TPFE or TPFE−siRNA complexes were observed at the sample-loading wells in bright-field (BF) images, which is in good agreement with the results of loading efficiency measurement (Figure 4a,b). In the presence of serum, 63% of naked siRNA was degraded within 15 min (lane 2; Figure 4c,d; see also Figure S2), whereas in the same condition, the siRNA in TPFE−siRNA complex remained (22% degradation, lane 3; Figure 4c,d). The 64% recovery of siRNA from the complex without serum exposure (Figure S2) indicates that the 22% loss of siRNA (lane 3) is mainly due to incomplete extraction of the remaining siRNA in the TPFE− siRNA complex. This result indicates that the sub-micrometersized TPFE−siRNA complexes efficiently protect siRNA from enzymatic degradation. siRNA Delivery and Cytotoxicity of TPFE−siRNA Complexes in Vitro. The efficiency of in vitro siRNA delivery of TPFE−siRNA complexes was evaluated by the knockdown of EGFP mRNA expression as measured by quantitative real-

noted that the R value of 5 showed the highest transfection efficiency of plasmid DNA,9,22 but resulted in a lack of significant knockdown efficiency in the case of siRNA. Taken together with the results of binding efficiency (Figure 3a,b), the low knockdown efficiency at R = 5 is attributed to the low loading amount of siRNA as well as the low cell internalization of siRNA because of the formation of the large aggregates of TPFE−siRNA complexes. It is known that siRNA is a linear oligonucleotide and exposes higher surface charge than that of cyclic plasmid DNA.34 Therefore, a higher amount of the cationic TPFE is necessary to shield the negative charge of siRNA for internalization into cells. Interestingly, at the R value of 10, TPFE−siGFP complex showed no significant knockdown, whereas 55% knockdown was observed by treatment of TPFE−Stealth siGFP complex. This result is in good agreement with the fact that the loading amount of TPFE− siRNA (R = 10) is lower than that of TPFE−siRNA (R = 20), but TPFE−siRNA can deliver enough Stealth siRNA into cells because of the high stability of Stealth siRNA under the physiological condition. The high knockdown efficiency notwithstanding, Stealth siRNA often causes problems in D

DOI: 10.1021/acsami.8b01869 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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terms of clearance in vivo because of its stability against enzymatic degradation. For a further practical application, our results clearly suggest that TPFE can effectively internalize the natural siRNA into cells and achieve high knockdown efficiency. According to the overall results, we concluded that the R value of 20 showed the most suitable ratio for siRNA delivery for TPFE both in the knockdown efficiency as well as in size for cellular internalization and hierarchical assembly. Cytotoxicity is an inevitable drawback for all of the delivery carriers at present because common cationic lipid-based carriers have dose-dependent cytotoxicity.35−37 Previous reports have already revealed that the TPFE−DNA complex in vitro22 and in vivo,9 and TPFE−siRNA in vivo,10 have no or low toxicity. Here, we performed quantitative evaluation of cytotoxicity of TPFE−siRNA complexes using MTT assay.38,39 TPFE−siRNA complexes were treated with 293-GFP cells at various concentrations ranging from 5.00 nM to 2.25 μM and the cells were incubated for 48 h. To reduce the effect of knockdown for cytotoxicity, both siGFP and siNEG were used. According to the fitting curves, the IC50 values of TPFE−siGFP and−siNEG complexes were determined as 710 and 690 μM of TPFE, respectively (Figure 5b). Through comparison of the dose of in vitro transfection (40 nM of TPFE), we conclude that TPFE−siRNA complexes do not have any cytotoxicity under the condition of therapeutic usage. In addition, we also reported that the fast clearance of TPFE after the release of siRNA led to low tissue toxicity in vivo.10 According to the results of DLS and STEM analyses (Figure 2), the watersoluble sub-10 nm-sized aggregates may undergo the fast clearance from lung after the release of siRNA,40 leading to the clearance by kidney.7 These results suggest that both the low cytotoxicity and the primary sub-10 nm aggregated structure gave the low tissue toxicity in vivo.10

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EXPERIMENTAL SECTION

Preparation of TPFE−siRNA Complexes. TPFE was synthesized by following the procedure reported previously.22 An EGFP-targeting siRNA, siGFP, and a nontargeting siRNA, siNEG (see also Table S1), were purchased from Hokkaido System Science Co., Ltd. Stealth siGFP (Stealth RNAi GFP Receptor Control) was purchased from Invitrogen. TPFE dissolved in 2 mM potassium chloride solution (pH 2.0) and siRNA dissolved in nuclease-free water were mixed to obtain a reagent-to-base pair ratio (R) of 1:50. The R value was calculated by dividing the nitrogen-to-phosphorus (N/P) ratio by 2.9,10 The mixture was incubated at room temperature for 5 min and then mixed with 10× phosphate buffered saline (PBS, pH 7.4; Gibco) before injection. Cell Line. EGFP-overexpressing human embryonic kidney cell line (293-GFP) was purchased from GenTarget Inc. (San Diego, CA) and maintained in D-MEM with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. DLS and LD Analysis. To evaluate the sizes of agglutination, DLS and LD analyses were conducted. DLS measurement was performed on a Malvern Zetasizer Nano ZS equipped with a He−Ne laser operating at 4 mW power and 633 nm wavelength, and a computercontrolled correlator at an accumulation angle of 173°. The measurement was performed at room temperature in a polystyrene or glass cuvette. The data were processed using Dispersion Technology Software version 4.10 to give Z-average particle size and polydispersity index values by cumulant analysis, and particle size distribution by CONTIN analysis.42,43 The DLS samples of TPFE− siRNA complexes were prepared as follows. Mouse serum was prepared just before use. TPFE−siRNA complex (R = 20) was prepared using 100 μg of siRNA and was incubated for 5 min at room temperature. LD measurement was performed in a Shimadzu Aggregates Sizer equipped with a semiconductor laser operating at 405 nm wavelength. The measurement was carried out at room temperature in a glass cuvette (the light path length is 1 mm). The data were processed using WingSALD bio software version 3.1.1 to give particle size distribution. LD samples of TPFE−siRNA complexes were prepared following the same procedure as that for DLS measurement, and then diluted by 20 times with PBS, because of the limit of concentration. Time-dependent change in size was also monitored by LD. STEM Imaging of TPFE−siRNA Complexes. STEM measurement as shown in Figure 2b was conducted on a JEOL JEM-2100F at 294 K with a spherical aberration coefficient Cs = 1.0 nm at an acceleration voltage of 200 kV under reduced pressure of 1.0 × 10−5 Pa in the sample column. The current density is ca. 0.5 pA·cm−2. The imaging instrument used an ultrascan charge-coupled device (CCD) camera (512 × 512 pixels). Five microliter of TPFE (50 μM) was deposited on a TEM copper mesh coated with a carbon film (Super Ultra High Resolution Carbon film, thickness