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Self-assembly of siRNA/PEG-b-catiomer at integer molar ratio into 100 nm-sized vesicular polyion complexes (siRNAsomes) for RNAi and codelivery of cargo macromolecules Beob Soo Kim, Sayan Chuanoi, Tomoya Suma, Yasutaka Anraku, Kotaro Hayashi, Mitsuru Naito, Hyun Jin Kim, Ick Chan Kwon, Kanjiro Miyata, Akihiro Kishimura, and Kazunori Kataoka J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13641 • Publication Date (Web): 07 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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Journal of the American Chemical Society

Self-assembly of siRNA/PEG-b-catiomer at integer molar ratio into 100 nmsized vesicular polyion complexes (siRNAsomes) for RNAi and codelivery of cargo macromolecules 1,2,a

Beob Soo Kim,

1,a

Sayan Chuanoi,

5

3

3

5

2

1,*

Mitsuru Naito, Hyun Jin Kim, Ick Chan Kwon, Kanjiro Miyata, Kazunori Kataoka, 1Department

4

Tomoya Suma, Yasutaka Anraku, Kotaro Hayashi,

6,7,*

Akihiro Kishimura,

4,8,*

of Materials Engineering, Graduate School of Engineering, The University of Tokyo,

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 2Center

for Theragnosis, Biomedical Research Institute, Korea Institute of Science and

Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea 3Department

of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1

Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 4Innovation

Center of NanoMedicne, Kawasaki Institute of Industrial Promotion, 3-25-14

Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan 5Center

for Disease Biology and Integrative Medicine, Graduate School of Medicine, The

University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 6Department

of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Moto-oka,

Nishi-ku, Fukuoka 819-0395, Japan 7Center

for Molecular Systems, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395,

Japan 8Policy Alternatives Research Institute, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo

113-0033, Japan a Equal

contribution

* Corresponding

authors

ACS Paragon Plus Environment

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Abstract Vesicular polyion complexes (PICs) were fabricated through self-assembly of rigid cylindrical molecules, small interfering RNAs (siRNAs), with flexible block catiomers of poly(ethylene glycol) (2 kDa) and cationic polyaspartamide derivative (70 units) bearing 5-aminopentyl side chain. 100 nm-sized siRNA-assembled vesicular PICs, termed siRNAsomes, were fabricated in specific mixing ranges between siRNA and block catiomer. The siRNAsome membrane was revealed to consist of PIC units fulfilling a simple molar ratio (1:2 or 2:3) of block catiomer and siRNA. These ratios correspond to the minimal integer molar ratio to maximally compensate the charge imbalance of PIC, because the numbers of charges per block catiomer and siRNA are +70 and –40, respectively. Accordingly, the ζ-potentials of siRNAsomes prepared at 1:2 and 2:3 were negative and positive, respectively. Cross-section transmission electron microscopic observation clarified that the membrane thicknesses of 1:2 and 2:3 siRNAsomes were 11.0 nm and 17.2 nm, respectively. Considering that a calculated long-axial length of siRNA is 5.9 nm, these thickness values correspond to the membrane models of two (11.8 nm) and three (17.7 nm) tandemly-aligned siRNAs associating with one and two block catiomers, respectively. For biological application, siRNAsomes were stabilized through membrane-cross-linking with glutaraldehyde. The positively charged and cross-linked siRNAsome facilitated siRNA internalization into cultured cancer cells, eliciting significant gene silencing with negligible cytotoxicity. The siRNAsome stably encapsulated dextran as a model cargo macromolecule in the cavity by simple vortex mixing. Confocal laser scanning microscopic observation displayed that both of the payloads were internalized together into cultured cells. These results demonstrate the potential of siRNAsomes as a versatile platform for codelivery of siRNA with other cargo macromolecules.


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INTRODUCTION Polymeric vesicles have recently gathered considerable attention because of their unique hollow architecture featuring tunable membranes. The vesicular membranes generate a specific compartment (or cavity) segregated from the outer milieu, allowing protection of fragile bioactive macromolecules (BMs), such as proteins and nucleic acids. Thus, polymeric vesicles have been engineered for cell or organelle mimicries, nano/micro-reactors, and delivery vehicles of pharmaceuticals.1–5 Importantly, vesicular membranes have a function as a versatile reservoir besides a simple protective barrier. In fact, amphiphilic block copolymer vesicles, namely polymersomes, can encompass hydrophobic low-molecular-weight drugs, such as anticancer agents, in their hydrophobic membrane with a high loading capacity, thereby utilized as a potential nanocarrier for cancer therapy.6,7 Moreover, hydrophobic proteins, e.g., channel proteins and enzymes, can also be embedded in the hydrophobic membrane for controlling the membrane permeability and the catalytic reactions, respectively.8–10 However, efficient and stable embedding of BMs, particularly strongly hydrophilic or charged BMs, in the vesicular membrane still remains to be a major challenge because embedding conditions are substantially restricted for such BMs due to their susceptibility to decomposition and denaturation, and further, an increase in the amount of embedded BMs more likely compromises the membrane integrity, destabilizing the vesicular structure. One sophisticated approach to solve the above issue is fabricating polymeric vesicles utilizing BMs as a self-assembling component rather than a simple inclusion. With regard to vesicular selfassembly of strongly charged macromolecules, we have reported polyion complex vesicles, termed PICsomes, which are assembled from an oppositely charged pair of block/block or block/homo ionomers through their electrostatic interactions.5,11–14 PICsomes are characterized to have a controllable size from 100 to several hundred nanometer with a narrow size distribution, and further, can embed more than several thousand ionomers in the PIC membrane.15 Moreover, both stability and membrane permeability of PICsomes are tunable based on covalent cross-linking degree of vesicular membrane, which is advantageous for fabrication of semipermeable nano/micro-reactors as well as delivery of BMs under in vivo conditions.16–18 Thus, it is quite plausible to utilize the PICsome methodology for the vesicular self-assembly of charged BMs, allowing the formation of thermodynamically stable vesicles with an appreciably high loading efficiency of BMs. In this way, a huge amount of BMs can be concurrently delivered by single



vesicular vehicle while they are site-specifically released by proper tuning of the membrane stability. Additionally, the vesicular cavity can further encapsulate functional macromolecules, directed toward co-delivery of substances of different nature. A short double-stranded RNA molecule, i.e., small interfering RNA (siRNA),19,20 is utilized here as a negatively charged BM. siRNA has been highlighted over a recent decade because of its sequence-specific gene silencing activity, namely RNA interference (RNAi).21 The RNAi is the potent biological mechanism for selective degradation of a target messenger RNA in the mammalian cell cytoplasm and thus the RNAi trigger, i.e., siRNA, is expected as a next generation of versatile molecular target drug. However, the translation of siRNA to pharmaceuticals has been hampered by its fragile and negatively charged nature, which substantially limits the cellular internalization of intact siRNA and thus needs delivery vehicles.22–24 Apart from such biological aspects, it is worth noting that siRNA possesses a rigid cylindrical structure with a length of 5.9 nm.20 This structural characteristic may significantly affect the vesicular self-assembly with flexible block catiomers, as our previous study revealed that the siRNA rigidity dramatically suppressed the multimolecular self-assembly (or micelle formation) of siRNA in comparison with a flexible single-stranded RNA.25 Alternatively, a minimal ionomer pair between siRNA and block catiomer, termed unit polyion complex (uPIC), was distinctly observed at a wide range of concentration.14,25,26 The present study, for the first time, fabricates the vesicular structures using strongly charged and rigid BM, i.e., siRNA, as a self-assembling membrane component (Fig. 1) and then extensively examines their structural characteristics, as well as siRNA delivery potential. As a counterpart block catiomer, poly(ethylene glycol)-block-poly[N-(5-aminopentyl]-α,β-aspartamide] (PEG-bP(Asp-AP)) is synthesized to provide PICs with a PEG weight fraction (fPEG) of less than 10% for vesicular formation.5,27 A polyaspartamide derivative, P(Asp-AP), is selected as a catiomer segment because our previous studies demonstrated that relatively long alkyl side chains in polyaspartamide were preferred for PICsome formation.28 The siRNA-based PICsome (siRNAsome) formation is verified through structural analyses in terms of size, surface charge, morphology, and ionomer composition. Ultimately, we demonstrate the potential of siRNAsomes for siRNA delivery to cultured cells, associated with the encapsulation of dextran as a model cargo macromolecule in the cavity, providing a versatile platform for co-delivery of siRNA with other hydrophilic BMs (Fig. 1).


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Figure 1. Schematic illustration of the formation of polyion complex vesicles from siRNAs and PEG-b-P(Asp-AP)s (siRNAsomes) and the encapsulation of cargo macromolecules in the cavity. RESULTS AND DISCUSSION Preparation and physicochemical characterization of PICs. A relatively long aliphatic spacer in a pendant group of cationic segment and a small fPEG in PICs (< 10%) are preferable for vesicular formation.28 Following these criteria, we synthesized a block catiomer, PEG-b-P(Asp-AP), with a Mn of PEG = 2000 and a DP of P(Asp-AP) = 70 for fabrication of siRNAsomes. Here, P(Asp-AP) bearing 5 carbon atoms in aliphatic spacer in the side chain was selected as a catiomer segment because a previous study demonstrated that the number of carbon atoms more than 4 allowed for the successful PICsome formation in a relatively wide range of fPEG, i.e., 6 < fPEG < 10%.28 Of note, this block catiomer is calculated to render the value of fPEG less than 10% for PICs prepared at residual molar ratios of amino groups in block catiomer to phosphate groups in siRNA (defined as N/Pfeed) < 5.8. Unlike our previous studies on PICsomes, no significant increase in scattered light intensity (SLI) was observed for a PIC sample prepared at N/Pfeed = 1 compared with a free PEGb-P(Asp-AP) sample, suggesting no perceptible formation of multimolecular PICs, such as micelles and vesicles, at this mixing ratio. Thus, N/Pfeed was systematically altered to discover feasible conditions for multimolecular PIC formation with an increased SLI. A considerable increase in SLI was observed at N/Pfeed ranging from 1.2 to 2.8 (Fig. S3). Size/polydispersity index



(PDI) and morphology of the PICs prepared in this region were then evaluated by dynamic light scattering (DLS) (Fig. 2 and Table S1) and transmission electron microscopy (TEM) (Fig. S4), respectively. A symmetric change at around N/Pfeed = 1.7 was observed for size and morphology of PICs; at N/Pfeed = 1.7 micrometer-sized PIC coacervates with a clear phase-segregated structure were visualized in the TEM image, whereas the PIC solution was turbid and thus unavailable for the DLS measurement; at N/Pfeed = 1.6 and 1.8 relatively large PICs with a size ranging between 250 and 300 nm were observed; at N/Pfeed = 1.4 and 2.2 PICs with a size of around 100 nm were clearly observed; at N/Pfeed = 1.2 and 2.6 mixtures of 100 nm-sized PICs and 30 nm-sized PICs were observed. These symmetric changes in size and morphology might be ascribed to varying surface potentials of PICs, as their -potential (ZP) values progressively increased with an increase in N/Pfeed and passed ZP = 0 at around N/Pfeed = 1.7 (Fig. 2). Higher absolute values in ZP might provide PICs with greater electrostatic repulsive forces to suppress secondary aggregation between PICs. Vesicle-like architectures were observed in the TEM images except for the PIC prepared at N/Pfeed = 1.7. Thus, we carefully validated the vesicular structures in the PIC samples with a relatively narrow PDI of 0.1 prepared at N/Pfeed = 1.4 and 2.2. Their cross-section TEM images clearly display the membrane structures with a diameter of ~100 nm (Fig. 3), demonstrating successful fabrication of vesicular PICs (siRNAsomes) from siRNAs and PEG-b-P(Asp-AP)s.

Figure 2. Changes in hydrodynamic diameter and ZP of PICs prepared from PEG-b-P(Asp-AP) and siRNA at varying N/Pfeed ratios, determined by DLS and ELS, respectively, at 0.25 mg/mL.


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Dashed line at N/P = 1.7 indicates a turbid solution point, at which the DLS and ZP measurements were not properly performed.

Figure 3. Cross-section TEM images of siRNAsomes prepared from PEG-b-P(Asp-AP) and siRNA at N/Pfeed = 1.4 (A) and N/Pfeed = 2.2 (B). Each PIC sample was cross-linked with GA at [GA]/[NH2] = 0.4 before TEM imaging. With regard to the ZP values, the formation of positively charged siRNAsomes at N/Pfeed > 1.7 is apparently reasonable because surplus PEG-b-catiomers were fed under these conditions. However, the siRNAsomes prepared at N/Pfeed = 1.2–1.6 presented considerably negative ZP values despite surplus PEG-b-catiomers still existing in the solution. Therefore, we assumed that all PEG-b-P(Asp-AP) molecules might not participate in sub-µm-sized PIC formation. To verify this assumption, the siRNAsomes prepared at N/Pfeed = 1.4 and 2.2 were further characterized as negatively and positively charged representatives, respectively. Here, siRNAsomes were prepared with Cy3-labeled PEG-b-P(Asp-AP) (PEG-b-P(Asp-AP)-Cy3) and then underwent the ultracentrifugation to separate siRNAsomes (or sub-µm-sized PICs) from the other fractions including free PEG-b-P(Asp-AP)-Cy3. Sedimentation of sample solutions dramatically decreased SLI of the supernatant, indicating successful separation of sub-µm-sized PICs from the sample solution. The quantities of PEG-b-P(Asp-AP)-Cy3 and siRNA remaining in the supernatant were determined by fluorospectrophotometer and UV spectrophotometer, respectively, and converted to the percentage to the initial quantities before ultracentrifugation (Table 1). At both N/Pfeed ratios, roughly half of PEG-b-P(Asp-AP)-Cy3 molecules remained in the supernatant without forming sub-µm-sized PICs. Based on the obtained values, effective N/P (N/Peffect) ratios of the sedimentate



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fraction (or siRNAsomes) were calculated to be 0.82 and 1.2 for N/Pfeed = 1.4 and 2.2, respectively (Table 1), consistent with the negative and positive ZPs, respectively. According to these N/Peffect values, a molar ratio of block catiomer and siRNA (catiomer:siRNA) contained in siRNAsomes was further estimated to be 1:2 and 2:3 for N/Pfeed = 1.4 and 2.2, respectively (Table 1). Table 1. Block catiomer and siRNA compositions in supernatant and sedimentate of PIC sample. N/Pfeed = 1.4

a b

N/Pfeed = 2.2

Supernatant

Sedimentate

Supernatant

Sedimentate

PEG-b-P(Asp-AP)a

47%

53%

59%

41%

siRNA a

10%

90%

22%

78%

N/Peffect b

0.82

1.2

catiomer:siRNA c

1:2

2:3

determined by spectrophotometric measurements of supernatants. defined as a residual molar ratio of primary amines in block catiomer to phosphates in siRNA contained in

sedimentate fraction and calculated by the formula: N/Pfeed × [% in PEG-b-P(Asp-AP)]/[% in siRNA]. c

estimated as a minimal integer ratio fulfilling the above N/Peffect.

A detailed structural analysis based on the cross-section TEM images (Fig. 3) provides additional information of the membrane structure in siRNAsomes. Considering that only PIC domains were stained in the cross-sections, the thicknesses of PIC layer in siRNAsomes were determined to be 11.0 ± 1.1 nm (n = 100 vesicles) at N/Pfeed = 1.4 and 17.2 ± 1.1 nm (n = 100 vesicles) at N/Pfeed = 2.2. These values are close to a double and triple of the siRNA length (5.9 nm in 21-mer/21-mer),20 respectively. Combined with the aforementioned catiomer:siRNA ratios, it is proposed that two and three siRNA molecules might be tandemly aligned with one and two PEG-b-P(Asp-AP)(s), respectively, in the PIC membrane as illustrated in Scheme 1. These tandem alignments might result from the uniform and regular packing of rigid siRNA molecules, which need to be ion-paired with P(Asp-AP) chains in the confined space of PIC layer sandwiched by PEG palisades for lamellar formation.


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Scheme 1. Schematic illustration of PIC membranes in two states of siRNAsomes. With regard to the minimal association units, their secondary associations above a critical association concentration (CAC) might generate multimolecular siRNAsomes as previously demonstrated for micellar and vesicular PICs.14,25 Thus, the secondary association manner of the minimal association units (or uPICs) was further validated for siRNAsome formation. The CAC values were first determined for siRNAsomes (N/Pfeed = 1.4 and 2.2) based on the change in size plotted against concentration (Fig. S5). Both PICs exhibited a clear increase in hydrodynamic diameter from a concentration of around 0.001 mg/mL and the increase in size leveled off at approximately 100 nm. These results are in good agreement with the secondary association behavior of uPICs toward multimolecular assemblies above the CAC. The smaller PICs (or uPICs) with a diameter of 6–7 nm observed at lower concentrations were further characterized by fluorescence correlation spectroscopy (FCS)25 to determine the association numbers of siRNA (ANsiRNA) and block catiomer (ANcatiomer) contained in a PIC, as summarized in Table 2. The obtained values at N/Pfeed = 1.4 were nicely correlated with the uPICs proposed in Scheme 1. On the other hand, the values obtained at N/Pfeed = 2.2 were apparently not matched with our expected values (i.e., ANsiRNA = 3 and ANcatiomer = 2). The non-integer ANsiRNA of 1.5, which was obtained as average, suggests that the uPIC samples might be a 1:1 mixture of single siRNA-loaded uPICs



and two siRNA-loaded uPICs, both of which were comprising a single PEG-b-catiomer (Scheme S1). The slightly smaller size of uPICs prepared at N/Pfeed = 2.2, compared with those prepared at N/Pfeed = 1.4, might be due to the presence of single pairs of siRNA and block catiomer. Thus, at N/Pfeed = 2.2, two states of uPICs (1 catiomer/1 siRNA and 1 catiomer/2 siRNAs) were likely complexed with each other above the CAC (Scheme S1), allowing for multimolecular associations toward the vesicular architecture. The observed self-assembling behavior of siRNAsome is apparently distinctive from that of PICsomes previously prepared from flexible block/homo (co)polymer pairs, which assembled into PICsomes through the charge neutralization at N/Pfeed = 1.0 and thus, the assembling behavior can be briefly described by fPEG.4,28 In siRNAsome formation, the structural characteristic of siRNA, i.e., monodispersed rigid cylindrical structure with negative charges presented at regular intervals, more likely hinders such simple (or complete) charge neutralization between siRNA and flexible block catiomers. Consequently, the self-assembling behavior of siRNAsome is apparently described by two factors; uPIC formation according to the minimal integer molar ratio accompanied by incomplete charge neutralization, and secondary association of uPICs according to their charged states, which affect the colloidal (or dispersion) stability of resulting multimolecular PICs.


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Table 2. Sizes of uPIC and association numbers of siRNA and catiomer in uPIC. N/Pfeed = 1.4

N/Pfeed = 2.2

Hydrodynamic diameter (nm)a

7.1

6.3

ANsiRNA b

2.0 ± 0.1

1.5 ± 0.1

ANcatiomer c

1.0 ± 0.1

1.1 ± 0.2

a determined

for Alexa488-siRNA at 10 nM by FCS.

b determined

for Alexa488-siRNA at 10 nM by FCS.

c determined

for PEG-b-P(Asp-AP)-Cy3 at 10 nM by FCS.

Stabilization of siRNAsomes for biological evaluation. The feasibility of siRNAsomes as an siRNA carrier was verified for the siRNAsomes prepared at N/Pfeed = 1.4 and 2.2 in the following biological evaluations. The composition determination indicates that siRNAsome formation should be equilibrated with free PEG-b-catiomers (or uPICs) in solution. This further indicates that siRNAsomes may be destabilized upon dilution under physiological conditions, presumably due to an equilibrium shift. To overcome this stability issue, siRNAsomes were stabilized by crosslinking the PIC membrane (or primary amines in catiomer segment) with glutaraldehyde (GA).29 Various molar mixing ratios of GA to amines in PEG-b-P(Asp-AP) ([GA]/[NH2]) of 0.0, 0.4, 0.8, and 1.0 were tested for cross-linking. The resulting PIC samples were characterized by DLS and ZP measurements (Table 3). For both siRNAsomes (N/Pfeed = 1.4 and 2.2), the ZP values were gradually decreased with an increase in [GA]/[NH2], suggesting that GA reacted with primary amines in the PIC membrane in a concentration-dependent manner. Whereas the initial size of siRNAsome (N/Pfeed = 1.4) was maintained after cross-linking, the siRNAsome (N/Pfeed = 2.2) showed a significant increase in size after cross-linking at [GA]/[NH2]  0.8. Considering that the ZP values of siRNAsome (N/Pfeed = 2.2) treated at [GA]/[NH2]  0.8 were close to the neutral, the size increase might be attributed to secondary aggregate formation between siRNAsomes with reduced electrostatic repulsive force. Indeed, larger aggregates derived from several vesicles were observed after cross-linking of siRNAsomes (N/Pfeed = 2.2) at [GA]/[NH2]  0.8 in TEM images (Fig. S6).



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Table 3. ZP, size, and PDI of cross-linked siRNAsomes at varying [GA]/[NH2]. Diameter (nm) N/Pfeed

1.4

2.2

a Samples b N/A

PDI

[GA]/[NH2]

ZP (mV)

without NaCl

with NaCl a

without NaCl

with NaCl a

0

–13.9

104

N/Ab

0.10

N/Ab

0.4

–17.6

106

103

0.11

0.11

0.8

–19.0

108

105

0.14

0.13

1.0

–21.0

111

110

0.16

0.10

0.12

N/Ab

0

17.8

104

N/Ab

0.4

9.8

101

102

0.12

0.11

0.8

3.1

161

168

0.20

0.14

1.0

0.6

184

179

0.19

0.25

(5 µM siRNA) were incubated in 150 mM NaCl solution at 37 °C for 2 days before DLS measurement.

indicates that the DLS measurement was not available because of high turbidity of sample solutions.

Next, stability of cross-linked siRNAsomes was examined by DLS in terms of size change after incubation at 37 °C for 2 days under a physiological salt condition (150 mM NaCl) (Table 3). The DLS results revealed that all cross-linked siRNAsomes maintained their initial size, demonstrating the significant stability of cross-linked siRNAsomes under the physiological salt condition. In contrast, the solution of non-cross-linked siRNAsomes became cloudy during incubation with 150 mM NaCl, indicating that non-cross-linked siRNAsomes formed aggregates in the salt solution, as previously observed.13 The serum tolerability of cross-linked siRNAsomes ([GA]/[NH2] ≥ 0.4) was further evaluated by FCS in terms of change in the diffusion coefficient (Fig. 4). Note that an FCS analysis was utilized to measure the diffusion coefficient of fluorescent nanoparticles (siRNAsomes prepared with Alexa488-siRNA) diluted in protein-contaminant solutions because of a better signal-to-noise ratio compared with DLS.30,31 No significant changes in diffusion coefficient were observed for all the cross-linked siRNAsomes incubated with 10% FBS for 48 h, demonstrating high tolerability of cross-linked siRNAsomes in the FBS solution. The enhanced colloidal stability of cross-linked siRNAsomes in physiological milieu was presumably ascribed to the formation of covalent cross-linking networks among catiomers, as well as the hydrated PEG palisades preventing nonspecific protein adsorption. Thus, the GA crosslinking of siRNAsomes was proven to be essential for biomedical applications.


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Figure 4. Serum tolerability of cross-linked siRNAsomes prepared at varying [GA]/[NH2]. Each PIC sample was prepared with Alexa488-siRNA and incubated with or without 10% FBS in 10 mM HEPES (pH 7.3) and 150 mM NaCl for 48 h at 37 °C. The diffusion coefficient was determined at 1.25 µM siRNA (125 nM Alexa488-siRNA) by FCS analysis. Results are expressed as mean ± SD (n = 10). siRNA delivery to cultured cancer cells. To evaluate the siRNA delivery potential, siRNAsomes were applied for a luciferase assay in cultured hepatoma cells expressing luciferase gene (Huh-7Luc). The siRNAsome samples were prepared from luciferase-targeted siRNA (siLuc) or nontargeted scramble siRNA (siScr), and incubated with the cells for 48 h. Then, the relative luminescence intensity of the cell lysates was recorded (Fig. 5A). Significant gene silencing was observed for the cross-linked siRNAsomes prepared from siLuc at N/Pfeed = 2.2 and [GA]/[NH2] = 0.4, whereas the others showed a negligible effect. No gene silencing of the cross-linked siRNAsomes prepared from siScr at N/Pfeed = 2.2 and [GA]/[NH2] = 0.4 confirms the sequencespecific gene silencing activity of the cross-linked siRNAsomes. To gain further insight to the significant gene silencing derived from the specific siRNAsome, the cellular uptake efficiency of siRNAsomes was evaluated by imaging cytometric analysis (Fig. 5B and Fig. S7), where siRNAsomes were prepared from Alexa488-siRNA and incubated with the cells for 48 h. The quantitative result displays a general tendency of higher fluorescence



intensities from the cells treated with cross-linked siRNAsomes compared with non-cross-linked siRNAsomes. This result indicates more efficient cellular uptake of cross-linked siRNAsomes, presumably due to their higher tolerability in serum-containing medium. Additionally, the crosslinked siRNAsomes at N/Pfeed = 2.2 elicited higher fluorescence intensities compared with those at N/Pfeed = 1.4, and further, cross-linked siRNAsomes at lower [GA]/[NH2] exhibited higher fluorescence intensity. These cellular uptake tendencies of cross-linked siRNAsomes are apparently correlated with their varying ZP values, as the higher ZP permitted the higher fluorescence intensity from the cells. Positively charged cross-linked siRNAsomes might more strongly bind to the negatively charged cellular surface, facilitating the adsorptive endocytosis. Notably, the cross-linked siRNAsomes at N/Pfeed = 2.2 and [GA]/[NH2] = 0.4 with high stability as well as relatively higher ZP achieved the most efficient cellular uptake in cultured cells, consistent with the higher gene silencing efficacy. Subcellular distribution of the cross-linked siRNAsomes (N/Pfeed = 2.2 and [GA]/[NH2] = 0.4) was further visualized by confocal laser scanning microscopy (CLSM). A major portion of siRNAsomes (or Alexa488-siRNAs) were apparently entrapped in late endosomes in 24-h transfection as many yellow pixels derived from colocalization of Alexa488-siRNAs (green) with late endosomes (red) were observed in the cells (Fig. S8A). Even though a number of yellow pixels still remained in the cells, a region of green pixels corresponding to non-colocalized Alexa488-siRNA with late endosomes were also observed in 24-h post-incubation after medium change (Fig. S8B), suggesting that a part of siRNAsomes were released from late endosomes to the cytoplasm to induce the significant gene silencing effect. This result also suggests that further functionalization of siRNAsomes with endosome-disrupting polymers32 more likely improves their gene silencing efficiency by facilitating the endosomal escape of siRNAsomes.


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Figure 5. (A) Relative luminescence intensity of Huh-7-Luc cells incubated with siRNAsome samples prepared from siLuc or siScr at 300 nM siRNA for 48 h. Relative luminescence intensities were obtained by normalizing to those from non-treated control cells. (B) Relative fluorescence intensity of Huh-7-Luc cells incubated with siRNAsome samples prepared from Alexa488-siRNA at 300 nM siRNA for 48 h. Relative fluorescence intensities were obtained by normalizing to non-



treated control cells. (C) Endogenous gene silencing efficiency of cross-linked siRNAsomes (N/Pfeed = 2.2 and [GA]/[NH2] = 0.4) in cultured A549 cells, determined by real-time qRT-PCR. The cells were incubated with samples containing siVEGF or siScr at 100 and 300 nM siRNA for 48 h. The amount of VEGF mRNA was normalized to that of GAPDH mRNA from non-treated cells. All results are expressed as mean ± SD (n = 4; **p < 0.01). One question in the correlation between gene silencing and cellular uptake profiles is why the cross-linked siRNAsomes at N/Pfeed = 2.2 and [GA]/[NH2] = 0.8 and 1.0 had almost no gene silencing activity (Fig. 5A) despite their modest cellular uptake efficiency (Fig. 5B). We assumed that the higher [GA]/[NH2] > 0.4 might result in overstabilization of cross-linked siRNAsomes, associated with inefficient siRNA release in the cell cytoplasm. To verify this assumption, we evaluated the siRNA releasability of cross-linked siRNAsomes through an exchange reaction with poly(aspartic acid) (PAsp) (Fig. S9). Of note, this experiment was designed following the previous studies that siRNAs are mainly released from PICs through the counter polyanion exchange with negatively charged biological components.31,33 The siRNAsomes (N/Pfeed = 2.2) with the higher degree of cross-linking considerably decreased the amount of released siRNA in the presence of PAsp, indicating the poor siRNA-releasability. These results suggest that the degree of crosslinking should be optimized for both significant stability of siRNAsomes in physiological milieu and smooth release of siRNA within the cells. The obtained results demonstrate that the crosslinked siRNAsomes prepared at N/Pfeed = 2.2 and [GA]/[NH2] = 0.4 had an optimized balance of stability and releasability. It should be noted that this cross-linked siRNAsome elicited negligible cytotoxicity in cultured Huh-7-Luc cells at siRNA concentrations of 100, 300, and 500 nM (Fig. S10). The optimized cross-linked siRNAsomes (N/Pfeed = 2.2 and [GA]/[NH2] = 0.4) were further applied to silence vascular endothelial growth factor (VEGF) gene in cultured human lung carcinoma (A549) cells. VEGF is a key mediator of angiogenesis essential for the growth of various tumors, thereby considered as a potential target of RNAi-based cancer therapy.34,35 VEGF mRNA levels were measured by real-time qRT-PCR after 48-h incubation (Fig. 5C). The crosslinked siRNAsomes equipped with siVEGF at siRNA concentrations of 100 and 300 nM significantly reduced the level of VEGF mRNA to 60% and 50%, respectively. In contrast, no VEGF mRNA reduction was observed in the cells treated with the cross-linked siRNAsomes


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prepared from siScr. The obtained result reveals the endogenous gene silencing ability of crosslinked siRNAsomes, demonstrating their potential utility for antiangiogenic cancer therapy. Encapsulation of hydrophilic macromolecules into siRNAsomes and their cellular delivery. When internalized by cells, exogenous BMs, such as enzymes and antibodies, can greatly contribute to various therapeutic modalities.36 However, such BMs cannot efficiently enter cells because of their hydrophilicity and modest surface charge.37 One attractive feature of vesicular nanoparticles is their capacity to encapsulate hydrophilic macromolecules within the cavity for their delivery to the target cell. Particularly, PICsomes have been demonstrated to physically encapsulate hydrophilic macromolecules through fragmentation to uPICs and re-association to PICsomes by simple vortex mixing in aqueous media.14,17,18 Thus, an encapsulation capacity of siRNAsomes was examined using tetramethylrhodamine isothiocyanate labeled-dextran (TRITCDex) (MW ~70,000) as a model hydrophilic macromolecule. TRITC-Dex was added to an aqueous solution of preformed non-cross-linked siRNAsomes (N/Pfeed = 2.2), followed by vortex mixing. Then, siRNAsomes were cross-linked at [GA]/[NH2] = 0.4. The cross-linked sample solution was ultrafiltrated to remove free TRITC-Dex molecules, as well as GA and glycine. The resulting solution was divided into five samples: the first was simply diluted with 10 mM HEPES buffer (pH 7.3), the second was incubated with 10 mM HEPES buffer (pH 7.3) containing 150 mM NaCl and 10% FBS at 37 °C for 48 h, and the others were incubated with 10 mM HEPES buffer (pH 7.3) containing PAsp at C/P = 0.25, 1.50, or 15.0 at 37 °C for 1 h to examine the TRITC-Dex releasability of cross-linked siRNAsomes. The encapsulation of TRITC-Dex into cross-linked siRNAsomes was verified in terms of change in the autocorrelation curve of TRITC-Dex obtained from the FCS analysis (Fig. 6A). The autocorrelation curve of Dex@siRNAsome in buffer was dramatically shifted toward larger relaxation time compared with that of free TRITC-Dex, consistent with encapsulation of TRITC-Dex in the cross-linked siRNAsomes. Notably, the similar autocorrelation curve was obtained even after 48-h incubation with 10% FBS, confirming the stable encapsulation capacity of cross-linked siRNAsomes in the physiological milieu. The diffusion coefficients of encapsulated TRITC-Dex were determined to be 2.8 ± 0.5 and 2.6 ± 0.8 μm2/sec for Dex@siRNAsome in buffer and in 10% FBS, respectively. These values are in good agreement with those of Alexa488-siRNA-loaded siRNAsomes (Fig. 4). Of note, 0.9% of dextran initially fed into the solution was entrapped in Dex@siRNAsome, as estimated from the change in



TRITC-Dex concentration before and after encapsulation and purification. We further verified whether the encapsulated TRITC-Dex was released from Dex@siRNAsome accompanied by siRNA release. Thus, Dex@siRNAsomes were incubated with PAsp at varying residual molar ratios of carboxyl groups on PAsp to phosphate groups on siRNA (C/P) and the change in autocorrelation curve was monitored as an indicator of TRITC-Dex release (Fig. 6A). The autocorrelation curve was gradually shifted toward shorter relaxation time with an increase in C/P and coincided with that of free TRITC-Dex at C/P = 15, demonstrating the significant TRITC-Dex releasability of Dex@siRNAsomes. It should be noted that the incubation of siRNAsomes with PAsp resulted in structural changes of the vesicular architectures in the DLS analyses (Fig. S11). At C/P = 0.25, the SLI and size of cross-linked siRNAsomes were modestly increased, suggesting that a few amount of negatively charged PAsp might be adsorbed on the positively charged crosslinked siRNAsomes to induce their secondary aggregate formation, as observed in the crosslinking at N/Pfeed = 2.2 and [GA]/[NH2] = 0.8 and 1.0 (Fig. S6). Of note, the result obtained at C/P = 0.25 apparently does not match between FCS (i.e., almost no change in the autocorrelation curve in comparison with C/P = 0) and DLS (i.e., increase in SLI and size in comparison with C/P = 0), which should be due to the varying sample concentrations (115 nM siRNA in FCS vs. 2.5 µM siRNA in DLS). The much higher sample concentration in DLS should be more favorable for the secondary aggregate formation. In contrast, the increase in C/P from 1.5 to 15 resulted in the considerable decreases in SLI and size, suggesting that an excess amount of PAsp induced the structure change of cross-linked siRNAsomes into smaller PIC aggregates comprising PAsp. Next, the cellular delivery of TRITC-Dex by siRNAsomes was evaluated by a flow cytometric analysis for cultured A549 after 6-h incubation with Dex@siRNAsomes (Fig. 6B). The relative mean fluorescence intensities from the cells were determined to be 1.0, 1.3, and 3.3 for non-treated cells, free TRITC-Dex-treated cells, and Dex@siRNAsome-treated cells. Thus, the cells treated with Dex@siRNAsomes exhibited an 8-fold increase in cellular uptake efficiency of TRITC-Dex. This result demonstrates that the cross-linked siRNAsomes significantly facilitated cellular internalization of TRITC-Dex, a naked form of which was not efficiently uptaken by cells. To further confirm the simultaneous cellular delivery of TRITC-Dex with siRNA, the intracellular trafficking of Dex@siRNAsomes prepared from Alexa488-siRNA in cultured A549 cells was observed by CLSM (Fig. 6C). After 8-h incubation, a large fraction of yellow dots was observed in the cell interior (Fig. 6C-i), indicating that the majority of TRITC-Dex (green) was colocalized


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with Alexa488-siRNA (red) in the cells presumably due to their encapsulation in siRNAsomes. The fraction of yellow dots was progressively decreased with time (Fig. 6C-ii), and green and red dots were apparently separated after 48-h incubation (Fig. 6C-iii). This time-dependent decrease in colocalization of TRITC-Dex and Alexa488-siRNA was statistically analyzed using ImageJ software, based on the calculation of Pearson’s and Spearman's correlation coefficients between the green and red pixels in the CLSM images.38 Of note, both correlation coefficients are ranging between −1 and +1, where 0 indicates that there is no discernable correlation and −1 and +1 mean strong negative and positive correlations, respectively. Pearson’s correlation coefficients were calculated to be 0.64, 0.31, and 0.20 for 8 h, 24 h, and 48 h, respectively, and Spearman’s correlation coefficients were 0.58, 0.14, and 0.03 for 8 h, 24 h, and 48 h, respectively. Both correlation coefficients reveal that a strong positive correlation in the intracellular distribution of TRITC-Dex and Alexa488-siRNA at the initial stage was gradually lost with an increase in incubation time. These results indicate that cross-linked siRNAsomes loaded with Dex and siRNA were stably internalized into the cultured cells and then these payloads were gradually separated in the cells with the increase in incubation time. Taken together with the results from the siRNA and TRITC-Dex release assays, it is postulated that the cross-linked siRNAsomes encountered with polyanionic species in the cells should collapse to change their structure with releasing both payloads of siRNA and TRITC-Dex. Our previous studies have demonstrated stable encapsulation of active enzymes, such as β-galactosidase17 and L-asparaginase,39 into PICsomes via vortex mixing and membrane cross-linking, similar to Dex@siRNAsomes. More recently, the central RNAi effector protein, Argonaute 2, has been simultaneously delivered with siRNA using cationic polyaspartamide derivatives for enhanced RNAi efficacy.40 Thus, siRNAsomes are envisioned to encapsulate such proteins for their codelivery with siRNA into the cytoplasm, directed toward the synergistic biological effects.



Figure 6. (A) Autocorrelation curves of TRITC-Dex determined by FCS. The FCS measurement was performed for TRITC-Dex-incorporated cross-linked siRNAsomes (N/Pfeed = 2.2 and [GA]/[NH2] = 0.4) in 10 mM HEPES buffer (pH 7.3) (red line), incubated with 150 mM NaCl and 10% FBS for 48 h (green dashed line), and incubated with PAsp (C/P = 0.25, 1.50, and 15.0) for 1 h (yellow, orange, and brown dashed line). Free TRITC-Dex molecules were also analyzed as a control. The concentrations of TRITC-Dex and siRNA in siRNAsome samples were set at 23 nM and 115 nM, respectively. (B) Cellular uptake of free TRITC-Dex and Dex@siRNAsomes in cultured A549 cells, determined by flow cytometric analyses. The cells were incubated with free TRITC-Dex (60 nM) or TRITC-Dex-loaded siRNAsomes (60 nM TRITC-Dex, 300 nM siRNA) for 6 h. Results are expressed as mean ± SD (n = 4; ****p < 0.0001). (C) CLSM images of A549 cells incubated with Dex@siRNAsomes (60 nM TRITC-Dex, 300 nM Alexa488-siRNA). The cells were incubated with Dex@siRNAsomes for 8 h and washed with PBS, followed by no additional incubation (i), 24-h incubation (ii), and 48-h incubation (iii). Alexa488-siRNA and TRITC-Dex are shown in green and red, respectively. The nuclei were stained with Hoechst 33342. Inset is a magnified image at each white dashed square.


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CONCLUSIONS The present study demonstrated the formation of the monodispersed 100 nm-sized polymeric vesicles comprising siRNA-intercalated membrane, termed siRNAsomes, for the first time. The siRNAsomes were successfully fabricated through PIC formation between PEG-b-catiomer and siRNA at non-stoichiometric charge ratios. Interestingly, two states of siRNAsomes were clearly observed in terms of surface charge (or ZP) and membrane thickness. The detailed structural analyses revealed the integer molar ratio-based minimal association unit in each siRNAsome, i.e., 1 catiomer and 2 siRNAs at N/Pfeed = 1.4; and 2 catiomers and 3 siRNAs at N/Pfeed = 2.2, which reasonably explained the sign of ZP (negative or positive) and the membrane thickness (thinner or thicker) of the corresponding siRNAsomes. Thus, as for the PICs prepared from a pair of monodispersed and rigid macromolecule and flexible block copolymer, the resulting structure may be directed according to an integer molar ratio of the charged pair. This insight is beneficial for the design of nanoarchitectures using biomacromolecules with a precisely regulated higher ordered structure,

including

oligonucleotide

aptamers

and

proteins,

besides

double-stranded

oligonucleotides. With respect to biological applications, the cross-linked siRNAsomes with positive surface charge exerted the high stability in salt and serum-containing milieu and elicited the significant endogenous gene silencing without cytotoxicity in cultured cancer cells, presumably due to the efficient cellular uptake and effective siRNA release. Ultimately, siRNAsomes were demonstrated to entrap hydrophilic macromolecules (TRITC-Dex) in their cavity, stably maintain them under a physiological condition, and simultaneously deliver them with siRNAs into cultured cells. Because of these unique characteristics, siRNAsomes may be useful for loading various types of BMs with different modes of action both in the membrane structure and in the inner cavity, and can work as multifunctional nanocarriers for various pharmaceutical applications, especially oligonucleotide-based multimodal therapeutics and theranostics. ASSOCIATED CONTENT Supporting Information Experimental section, schematic illustration of expected uPIC structure (N/Pfeed = 2.2) (Scheme S1), DLS results of PICs prepared at varying N/Pfeed (Table S1), 1H NMR spectrum (Fig. S1) and



GPC chart (Fig. S2) of PEG-b-P(Asp-C5), SLS results (Fig. S3) and TEM images (Fig. S4) of PICs prepared at varying N/Pfeed, sizes of PICs prepared at varying concentrations (Fig. S5), TEM images of cross-linked siRNAsomes (N/Pfeed = 2.2) at [GA]/[NH2] = 0.8 and 1.0 (Fig. S6), fluorescence micrographs obtained from imaging cytometry (Fig. S7) and CLSM observation (Fig. S8), results of siRNA release assay (Fig. S9), cell viability assay (Fig. S10), and the structural change of siRNAsomes in the presence of PAsp (Fig. S11). These materials are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors [email protected] [email protected] [email protected] Author Contributions B.S.K. and S.C. contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This research was financially supported in part by the Center of Innovation (COI) Program from the Japan Science and Technology Agency (JST), the Grant-in-Aid for Scientific Research (KAKENHI Grant Numbers: 25000006 to K.K., 23685037, 26288082, JP17K20109 to A.K., and 25282141, 17H02098, 18K19900 to K.M.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, Global Innovative Research Center (GiRC) Project (2012K1A1A2A01055811) of National Research Foundation (NRF) of Korea and Intramural Research Program KIST, the research grant from the association for the progress of new chemistry (to A.K.), and a grant JSPS Core-to-Core Program, A. Advanced Research Networks. We are grateful to Dr. S. Fukuda, The University of Tokyo Hospital, Mr. H. Hoshi and the Research Hub for Advanced Nano Characterization at The University of Tokyo for their valuable support in the TEM measurements.


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Table of Contents


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sized vesicular polyion complexes (siRNAsomes) - American

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