Systemic Delivery of Folate-PEG siRNA Lipopolyplexes with

Aug 3, 2017 - Protection of small interfering RNA (siRNA) against degradation and targeted delivery across the plasma and endosomal membranes to the f...
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Systemic Delivery of Folate-PEG siRNA Lipopolyplexes with Enhanced Intracellular Stability for In Vivo Gene Silencing in Leukemia Dian-Jang Lee,†,‡ Eva Kessel,†,‡ Taavi Lehto,†,■ Xueying Liu,§ Naoto Yoshinaga,∥ Kar̈ t Padari,⊥ Ying-Chen Chen,# Susanne Kempter,¶ Satoshi Uchida,§,∥ Joachim O. Rad̈ ler,‡,¶ Margus Pooga,⊥ Ming-Thau Sheu,# Kazunori Kataoka,*,§,□ and Ernst Wagner*,†,‡ †

Department of Pharmacy and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 Munich, Germany ‡ Nanosystems Initiative Munich (NIM), Schellingstr. 4, 80799 Munich, Germany § Innovation Center of NanoMedicine (iCONM), Institute of Industry Promotion-Kawasaki, 3-25-14 Tonomachi, Kawasaki-ku, 210-0821 Kawasaki, Japan ∥ Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-8656 Tokyo, Japan ⊥ Institute of Molecular and Cell Biology and Institute of Technology, University of Tartu, 23 Riia Str., 51010 Tartu, Estonia # School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250, Wuxin St., 11031 Taipei, Taiwan ¶ Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, 80539 Munich, Germany □ Policy Alternatives Research Institute, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033 Tokyo, Japan S Supporting Information *

ABSTRACT: Protection of small interfering RNA (siRNA) against degradation and targeted delivery across the plasma and endosomal membranes to the final site of RNA interference (RNAi) are major aims for the development of siRNA therapeutics. Targeting for folate receptor (FR)expressing tumors, we optimized siRNA polyplexes by coformulating a folate-PEG-oligoaminoamide (for surface shielding and targeting) with one of three lipo-oligoaminoamides (optionally tyrosine-modified, for optimizing stability and size) to generate ∼100 nm targeted lipopolyplexes (TLPs), which self-stabilize by cysteine disulfide cross-links. To better understand parameters for improved tumor-directed gene silencing, we analyzed intracellular distribution and siRNA release kinetics. FR-mediated endocytosis and endosomal escape of TLPs was confirmed by immuno-TEM. We monitored colocalization of TLPs with endosomes and lysosomes, and onset of siRNA release by time-lapse confocal microscopy; analyzed intracellular stability by FRET using double-labeled siRNA; and correlated results with knockdown of eGFPLuc protein and EG5 mRNA expression. The most potent formulation, TLP1, containing lipopolyplex-stabilizing tyrosine trimers, was found to unpack siRNA in sustained manner with up to 5-fold higher intracellular siRNA stability after 4 h compared to other TLPs. Unexpectedly, data indicated that intracellular siRNA stability instead of an early endosomal exit dominate as a deciding factor for silencing efficiency of TLPs. After i.v. administration in a subcutaneous leukemia mouse model, TLP1 exhibited ligand-dependent tumoral siRNA retention, resulting in 65% EG5 gene silencing at mRNA level without detectable adverse effects. In sum, tyrosine-modified TLP1 conveys superior protection of siRNA for an effective tumor-targeted delivery and RNAi in vivo.

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INTRODUCTION

systems, simultaneously targeting multiple genes, may be more suitable for the treatment of the majority of heterogeneous and often highly mutated cancers.2 However, siRNA application in

Small interfering RNA (siRNA) duplexes that enable downregulation of mRNA in an efficient and sequence-specific manner are expected to be excellent therapeutics for treating incurable diseases such as cancer.1−5 Single siRNA-containing gene silencing systems may be therapeutic for cancers with clear single driver gene mutations. Multiple siRNA-containing © XXXX American Chemical Society

Received: July 3, 2017 Revised: July 31, 2017 Published: August 3, 2017 A

DOI: 10.1021/acs.bioconjchem.7b00383 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 1. Targeted lipopolyplexes (TLPs) for siRNA delivery. (a) Structures of targeted PEGylated oligomer (356) and lipo-oligomers (454, 49, and 229). C: cysteine; K: lysine; Y: tyrosine; PEG: polyethylene glycol; Stp: succinoyl-tetraethylene pentamine; OleA: oleic acid; LinA: linoleic acid; K(and K[ refer to branchings by α- and ε-amino modification of lysines. Illustrations and TEM micrographs of (b) targeted PEGylated polyplexes (by folated-conjugated 356 and siRNA), lipopolyplexes (by oleic acid-modified 454 and siRNA), and (c) TLPs (by coformulations of targeted PEGylated oligomer, lipo-oligomer, and siRNA). TLP1: 356 + 454; TLP2: 356 + 49; TLP3: 356 + 229).

stabilization.26 Introducing cysteines as cross-linking motifs is a versatile way to increase siRNA binding, particle stability, and transfection efficiency.29,30 Polyethylene glycol (PEG) acts as a shielding domain and is attached to the folate as targeting ligand. As a result, this oligomer 356 can formulate siRNA as neutrally charged monomolecular nanoplexes with hydrodynamic diameter of ∼6 nm, successfully exhibiting folate receptor (FR)-specific cellular uptake and gene silencing in vitro and in vivo. However, some limitations remained to be overcome. Because of small particle size, 356 siRNA nanoplexes displayed a short circulation time of 15 min followed by renal excretion after systemic administration. Moreover, since 356 siRNA polyplexes lack robust endosomal escape capacity, modification of siRNA with an endosomolytic INF7 peptide was necessary for potent gene suppression.26 Combination with other cross-linking Stp oligomers without PEG did favorably increase particle size of formed targeted combinatorial polyplexes (TCPs). However, this did not resolve all the problems: still, INF7 peptide was required for endosomal escape and such siRNA polyplexes displayed limited in vivo stability.31 In the current work, we utilized sequence-defined lipo-oligomers29,32 to form a novel class of lipopolyplexes. These lipo-oligomers consist of three or four protonable Stp motifs as cationic backbone with terminal cysteines, and are conjugated with fatty acids via lysine linkers (Figure 1a and b). They possess the capacity for strong electrostatic siRNA binding and enhanced endosomal escape; compared to 356, they form larger siRNA nanoparticles (sizes in a range of 45− 200 nm).29,33,34 The additional introduction of hydrophobic fatty acids (oleic acids in oligomers 49 and 454; linoleic acids in 229) not only increases the stability of siRNA polyplexes by

clinics has remained challenging because of two major bottlenecks. First, siRNA molecules have limited stability in the biological fluids, since they are actively targeted by nucleases for degradation in extracellular and intracellular environment. Importantly, by using variety of chemical modifications to the siRNA these improvements in the medicinal chemistry of siRNA have allowed to largely overcome this stability issue.6,7 Second, the high charge distribution and molecular weight of siRNA impede their ability to cross the cell membrane.8 To improve the delivery of siRNA, they can be formulated with various polycations into polyplexes or related complexes4,9−14 and with cationic lipids into lipoplexes and other lipid nanoparticles (LNPs).5,15−21 Independent of the delivery method, most of the siRNA cargo will accumulate in the endosomal system where the majority of the siRNA is finally degraded in the lysosomes. Moreover, it has been recently demonstrated that recycling by exocytosis can further restrict the available dose and pharmacological activity of siRNA in the cells.22−24 Thus, it is widely recognized that potency of siRNA formulations will strongly rely on their ability to effectively escape from the endosomes.24−26 Fine-tuning of chemical structures of polycations can precisely improve the design of polyplexes for successful siRNA transfection.27−29 Recently, we described artificial oligoamino acids such as succinoyl-tetraethylene pentamine (Stp) which together with natural α-amino acids and solid phase-supported synthesis can be applied to develop monodisperse oligomers for siRNA delivery.30 Oligomer 356 (Figure 1a and b) contains a branched two-arm structure comprising eight Stp units providing the positive charge for siRNA binding and two cysteines for disulfide-based polyplex B

DOI: 10.1021/acs.bioconjchem.7b00383 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 1. TLP Formulationsa Formulation

PEG Oligomer

Lipo-Oligomer

TLP1 TLP2 TLP3

Targeted Folate-PEG24-K(Stp4-C)2 (356)

C-Y3-Stp2-[(OleA)2-K]K-Stp2-Y3-C (454) C-Stp2-[(OleA)2-K]K-Stp2-C (49) (LinA)2-K-C-Stp3-C (229)

NTLP1 NTLP2 NTLP3

Nontargeted A-PEG24-K(Stp4-C)2 (188)

C-Y3-Stp2-[(OleA)2-K]K-Stp2-Y3-C (454) C-Stp2-[(OleA)2-K]K-Stp2-C (49) (LinA)2-K-C-Stp3-C (229)

a

The sequence implies from C- terminal to N- terminal. A: alanine; C: cysteine; K: lysine; Y: tyrosine; PEG: polyethylene glycol; Stp: succinoyltetraethylene pentamine; OleA: oleic acid; LinA: linoleic acid; K(and K[ refer to branchings by α- and ε-amino modification of lysines.

conjugated Stp oligomer (356) form surface charge-neutral 6 nm siRNA nanoplexes (TEM images shown in Figure 1b).26 The bis(oleoyl) lipo-oligomer 454 forms positively charged siRNA particles with diameter of 100−130 nm (TEM images shown in Figure 1b) and very prominently enhanced siRNA binding (in comparison to 356) (Figures S1 and S2). Lipooligomer 454 is the structural analog of the T-shaped lipooligomer 49, with two terminal tyrosine trimer units increasing in vitro and in vivo stability of 454 siRNA polyplexes.32,34 The beneficial effect of tyrosines had been formerly established for siRNA PEI polyplexes.39 Thus, to optimize the physicochemical properties of siRNA polyplexes, we here combined 356 and 454 together by different [targeted PEG oligomer/lipooligomer] molar ratios to formulate siRNA as TLP1 formulation. Moreover, based on this strategy, as shown in Table 1 and Figure 1c, beside 454 two other lipo-oligomers, 49 (tyrosine-free analog) and 229 (an i-shaped, linoleic acid-based oligomer for effective siRNA transfection),29,30 were used for combination with targeted PEGylated oligomer 356 to prepare TLP2 and TLP3, respectively. The corresponding nontargeted NTLPs were formed using alanine-containing PEG oligomer 188 instead of 356. All oligomers contain terminal cysteines, which are essential for enabling polyplex-stabilizing cross-links through intermolecular disulfide formation.26,29 The protonable nitrogen/siRNA phosphate charge ratio (N/ P) was selected for polyplex preparation based on previous biophysical studies and reporter gene silencing assays.26,31,40 Stp-PEG oligomers 356 or 188 (with or without folate ligand) form approximately 6 nm small nanoplexes and neutral zeta potential 0 mV (±3 mV) for a broad range of N/P 3 to 40. Also, nanoparticle properties of 454 siRNA lipopolyplexes (∼100 nm, zeta potential around +19 to +27 mV) do not significantly change over a range of N/P 6 to 16.35,36 Apparently, the oligomer chemistry overrules the N/P charge ratio (with just a higher fraction of free unbound oligomer at higher N/P ratios). The high PEG content of 356 and 188 result in monomolecular decorated nucleic acid rod formation,26,41−43 while the lipo-oligomer glues together many siRNA molecules by hydrophobic core-stabilization. In the current study, N/P 16 was selected as an optimized ratio for polyplex preparation based on previous reporter gene silencing assay and in vivo studies. As measured by dynamic light scattering (DLS) (Table S1), the surface charge of TLP1 at N/ P 16 was strongly shifted from +19 mV (at the ratio of 0% PEG-oligomer: 100% lipo-oligomer) to +11.7 mV (at the ratio of 20%:80%) by addition of PEGylated 356. This provides a clear indication that PEGylation successfully shielded the surface charges in these TLPs. At the ratio of 30%:70%, TLP1 presented reduced zeta potential value (+7.0 mV) and diameter of ∼127 nm. Similarly, TLP2 displayed zeta potential

hydrophobic interactions, but also provides enhanced lytic activity toward endosomal lipid membranes.29,32 Further incorporation of terminal tyrosine trimers in the case of oligomer 454 results in higher siRNA polyplex stability and prolonged systemic distribution in vivo.32 Nevertheless, 49, 454, and 229 siRNA polyplexes still have their drawbacks; these polyplexes exhibit high positive charge (+21−28 mV),29,34−36 which are prone to recognition by mononuclear phagocyte system (MPS) upon systemic delivery.37 Also, such nontargeted lipopolyplexes induce negligible transfection efficiency in FRpositive tumors,35 and it would be difficult to minimize unintended side effects. In the current study, we coformulated siRNA with targeted PEGylated oligomer 356 and one of the three lipo-oligomers 49, 454, or 229. For each of these targeted lipoplexes (TLPs) (Figure 1c), nanoparticle characteristics were controlled by the ratios of siRNA, targeted PEG oligomer 356, and the respective lipo-oligomer. The tyrosine-modified oleic acid-based TLP1 was found to show the most potent gene silencing effect. Hence, it was expected to possess the best endosomal escape capability. Surprisingly, the translocation profiles of siRNA from late endosomes to cytosol indicated that the tyrosine-free formulations, TLP2 and TLP3, underwent earlier endosomal exit than TLP1. While the physicochemical characterization (zeta potential and size) and FR-mediated uptake of the TLPs are the same, the intracellular siRNA stability differed remarkably. As evaluated by fluorescence resonance energy transfer (FRET) analysis, siRNA remained largely intact in TLP1 at 4 h after transfection, which significantly outperformed the other TLPs. Encouragingly, followed by i.v. administration, TLP1 managed to downregulate distant FR-directed tumoral EG5 gene expression by 65% in a mouse leukemia model. The present data demonstrate the power of sequence-defined chemistry to regulate key delivery domains in polyplexes toward a higher efficacy. Taken together, TLP1 with addition of tyrosine trimers revealed higher transfection efficiency than those tyrosine-free TLPs, mainly due to a more durable intracellular siRNA stability than early endosomal escape capacity. These novel mechanistic findings widen our knowledge on the requirements for efficient siRNA delivery,38 and provide guidance for the further design of cytosolic siRNA carriers.

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RESULTS AND DISCUSSION Preparation of Targeted Lipopolyplexes (TLPs) and Evaluation of Their Physicochemical Properties. To modulate the composition of targeted lipopolyplexes (TLPs) for siRNA delivery, we chose four oligomers (Figure 1a) from our recently established library of sequence-defined oligomers26,29,30,32 as a starting point. The folate-PEGC

DOI: 10.1021/acs.bioconjchem.7b00383 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. Folate receptor (FR)-mediated uptake and intracellular distribution of TLPs. (a) The cellular internalization capacity of TLPs was evaluated by comparing the uptake levels of siRNA polyplexes in FR-overexpressing (L1210, M109, and KB) vs FR-deficient (MCF-7) cells. siRNA polyplexes were prepared at N/P 16 with Cy5-labeled siRNA. NTLP1, nontargeted 188 + 454 lipopolyplexes. The cells were incubated with siRNA polyplexes at 37 °C for 4 h before flow cytometric measurements. The amount of Cy5-positive cells was analyzed through excitation of the dye at 635 nm and detection of emission at 665/20 nm. (b−f) Immuno-TEM of FR-mediated endocytic pathway for TLP1 in FR-overexpressing KB cells. TLP1 was formed at N/P 16 with biotinylated siRNA tagged with neutravidin-gold particles (10 nm). These gold nanoparticles were used for locating siRNA, each TLP contained ∼20 to ∼60 gold tags. FR was visualized with anti-FR-α antibodies and Protein G coupled to 6 nm gold particles (green arrowheads in the images or red dots in the scheme). (b) Association of siRNA polyplexes with the cell surface after incubation of cells with siRNA polyplexes for 15 min. (c) The higher-magnification image of the blue frame in (a) showed the association of siRNA polyplexes with FR. (d) The higher-magnification image of red frame in (a) showed membrane invagination. (e) The higher-magnification image of green frame in (d) indicated FR localizing in close proximity to siRNA polyplexes. (f) siRNA polyplexes localized inside the vesicular structure after 1 h. (g−j) Time-lapse confocal laser scanning microscopy (CLSM) for TLP1 transduction in KB cells. TLP1 was prepared at N/P 16 with AF 647-labeled siRNA (red). The nuclei (speckled blue pattern) were stained with Hoechst 33342. The yellow and gray frames were enlarged as (h) and (j), respectively. (g) Late endosomes (green) were visualized with transfected Rab7a-RFP (CellLight Late Endosomes-RFP baculovirus). (i) Lysosomes (green) are marked with transfected Lamp1-GFP (CellLight Lysosomes-GFP baculovirus). Labels are presented in artificial colors. D

DOI: 10.1021/acs.bioconjchem.7b00383 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry value of +11.3 mV and size of ∼90 nm (Table S2) at the ratio of 30% PEG-oligomer:70% lipo-oligomer, while TLP3 exhibited reduced surface charge (+9.2 mV) and size of ∼198 nm at the ratio of 20%:80% (Table S3). Consistent among the TLPs, the morphology of formulations assessed by transmission electron microscopy (TEM) indicated the TLPs were homogeneous spherical nanoparticles with size in a range similar to the DLS measurements (Figure 1c). A higher content of 40−70% or more PEGylated oligomer 356 resulted in increased particle heterogeneity and agglomeration as observed from the DLS results. Due to the intrinsically small size of polyplexes with oligomer 356 (due to the high PEG content) the size of TLPs was accordingly reduced, when the PEGylated oligomer 356 became dominant (∼90%). To evaluate the siRNA binding ability of TLPs at different mixing ratios, we performed gel shift assays (Figures S3, S4, S5). When the fraction of lipo-oligomers was increased in the TLPs, the siRNA binding was greatly potentiated. Notably, complete siRNA binding was observed between the ratio (PEG-oligomer/lipooligomer) of 10%:90% and 30%:70% for TLPs. Compared with 356 polyplexes, the results indicated that the presence of the lipo-oligomer in TLP formulations substantially enhanced siRNA binding efficiency, with the tyrosine-modified 454 in TLP1 providing best capacity to stabilize the siRNA polyplexes.32 In conclusion, by coformulating two functional oligomers, TLPs with different chemical motifs were generated: T-shaped oleic acid-based TLP1 with additional tyrosine trimers, oleic acid-based TLP2 without tyrosine, and i-shaped linoleic acid-based TLP3. The successful modification of polyplexes was accompanied by distinct changes in the physicochemical properties with tunable sizes, surface shielding, and improved siRNA binding. According to these results, optimized molar ratios of PEG-oligomer: lipo-oligomer (30%:70% for TLP1 and TLP2; 20%:80% for TLP3) were used for the following in vitro and in vivo studies. Receptor-Mediated Internalization and Intracellular Distribution of TLPs. Cellular entry of siRNA carriers is one limiting factor for their therapeutic effectiveness, and folate has emerged as a notable targeting ligand capable of potent interaction with folate receptor (FR)-expressing tumor cells with high affinity (Kd: 10−9−10−10 M).31,44−47 To verify targeting capacity of the folate-linked TLPs, fluorescent Cy5labeled siRNA was exploited to study the cellular internalization profile by flow cytometry. The FR-rich murine lymphocytic leukemia suspension cell line L1210, the FR-rich adherent tumor cell lines M109 (murine lung carcinoma) and KB (human cervical carcinoma), and the FR-deficient cell line MCF-7 (human mammary adenocarcinoma)31,48−50 were subjected for the following experiments. Cell cultures were treated with TLPs at N/P 16 for 4 h at 37 °C, and then the uptake levels of TLPs were analyzed by flow cytometry (Figure 2a). All the TLP formulations exhibited strong cellular internalization in FR-rich cells. The uptake levels for the TLPs in L1210 cells were similar (91−93% positive cells), which were higher than the levels in M109 cells (76− 81% positive cells). On the contrary, all the TLP formulations barely entered FR-deficient MCF-7 cells. For control studies, alanine-substituted oligomer 188 and previously stated lipooligomers were coformulated as nontargeted lipopolyplexes (NTLPs) (Table 1). In KB cells, the level of TLP1 (88% positive cells) was comparable with 356 polyplexes (91% positive cells), while NTLP1 mediated insufficient cellular uptake. In short, these results gave evidence for internalization

of TLPs in a FR-specific manner, and the folate conjugates were required for association with the target tumor cells. The crucial question about the membrane association, translocation into cells, and the following trafficking of TLPs remained obscure. A powerful tool to elucidate the internalization mechanism of TLPs is TEM,51 which has been employed to examine the cell entry processes and intracellular distribution of various macromolecules and particles.52,53 Nevertheless, among these, few studies have focused on the targeted delivery of siRNA.22,31 To locate the receptor in relation to the plasma membrane and cellular organelles, the primary antibodies against FR were visualized with Protein Ggold conjugates (6 nm in diameter). To map the siRNA molecules, biotinylated siRNA was labeled with neutravidingold conjugate (10 nm in diameter). As demonstrated in Figure 2b, after incubating FR-positive KB cells with 10 nm gold labelcontaining TLP1 for 15 min, siRNA polyplexes were detected prominently as electron dense particles and associated quickly with the cell surface. In the higher-magnification image, siRNA polyplexes seemed to be in close contact with the FR (6 nm gold tags indicated by arrowheads in Figures 2c and S6). Subsequently, the colocalization of siRNA polyplexes and the FR (indicated by red dots in the scheme of Figure 2d and arrowheads in the enlarged image as Figure 2e) induced morphological changes in the plasma membrane, leading to the formation of membrane invaginations. Later at 1 h, siRNA polyplexes shifted deeper into the cytoplasm, being finally fully engulfed by the cell into vesicles (Figure 2f), which resembled the structures of endolysosomal pathway. Correspondingly, other TLPs also followed the same FR-specific uptake pattern (Figure S7). Besides, nontargeted NTLP1 were very rarely detected at the cell surface (data not shown), and this correlated well with the inefficient uptake level measured by flow cytometry in KB cells (Figure 2a). Additionally, when receptors were saturated with excess folate, drastically decreased cellular association of TLP1 with KB cells was observed by confocal laser scanning microscopy (CLSM) (Figure S8). As a next step, to further differentiate the involvement of endolysosomal compartments in the transport of TLPs, and to gain more detailed insight into the dynamics of TLPs, we formulated TLP1 with AF 647-labeled siRNA and performed time-lapse CLSM with cultured KB cells. We labeled late endosomes by baculoviral transduction with Rab-7a-RFP (CellLight Late Endosomes-RFP), and lysosomes with lysosomal associated membrane protein 1 (Lamp1)-GFP (CellLight Lysosomes-GFP). Within 1 h (Figure 2g), AF 647-labeled siRNA (red) polyplexes were gradually associated with late endosomes (green), and the colocalization of siRNA and late endosomes was obvious after incubation for 1 and 3 h. As shown in the enlarged image (Figure 2h), the overlap of two signals suggested that TLP1 was entrapped in late endosomes. Between 3 and 4 h, the association with late endosomes diminished; expectedly, at the same time, the colocalization of siRNA polyplexes and lysosomes (green) became excessive (Figure 2i). As shown in Figure 2j, the majority of siRNA polyplexes were found to be inside lysosomes at 4 h. Hence, these findings validated that TLPs were capable of efficient tumor cell-specific uptake, and a particular emphasis is placed on the intracellular distribution of TLPs during FRmediated endocytosis. FR appeared to serve as a docking point for TLPs, and the binding of TLPs to receptors shortly induced internalization of siRNA polyplexes within 15 min. Afterward, E

DOI: 10.1021/acs.bioconjchem.7b00383 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 3. Endosomal escape and gene silencing of siRNA-containing TLPs. (a−d) Morphological changes in TLP1-containing vesicles in FRoverexpressing KB cells at 4 h monitored by immuno-TEM. TLP1 was formed at N/P 16 with biotinylated siRNA tagged with neutravidin-gold particle (10 nm). These gold nanoparticles were used for locating siRNA, each TLP contained ∼20 to ∼60 gold tags. FR was visualized after cell culture with anti-FR-α antibodies and Protein G coupled to 6 nm gold particles (green arrowheads in the images). Discontinuous membrane of some endosomes resulted in endosomal escape of the siRNA molecules. (a) and (c) were accompanied by higher-magnification images, (b) and (d), respectively. (e) Reconstructed KB cell showing the distribution of siRNA, late endosomes, and lysosomes based on CLSM. KB cells were incubated with TLP1 (N/P 16) containing AF 647-labeled siRNA (red) for 4 h. Late endosomes (green) were visualized with transfected Rab7a-RFP (CellLight Late Endosomes-RFP baculovirus). Lysosomes (green) were marked with transfected Lamp1-GFP (CellLight Lysosomes-GFP baculovirus). Labels are presented in artificial colors. (f) The scheme is included for interpreting the results in (e). (g) Gene silencing efficiency of TLPs in KB cells expressing eGFPLuc fusion protein (KB/eGFPLuc cells) measured by luminometer. The siRNA polyplexes were prepared at N/P 16 with different siRNA sequences: eGFP-targeted siRNA (siGFP) and control siRNA (siCtrl). After incubation of TLPs for 45 min, the luciferase expression was analyzed at 48 h, and presented as percentage in comparison to untreated control cells.

polyplexes is one of the most important characteristics because it might affect uptake efficacy, kinetics, and internalization mechanisms.57 The size limit for nanoparticles to undergo receptor-mediated endocytosis is about 100−200 nm.58,59 Shape can also affect the cellular uptake of nanoparticles; for example, spherical particles are taken up by cells more efficiently compared with the rod-shaped particles.57 siRNA Release and Reporter Gene Silencing by TLPs. As most of TLPs retained their dense packing inside

at 1−3 h, late endosomes most often governed the transduction of TLP1. Then the vesicles trafficked through the endolysosomal pathway experiencing a gradual drop of pH, and siRNA polyplexes were finally targeted to lysosomes. Specifically, it has been reported that L1210 cells overexpress FR-α,54−56 and γconjugates (i.e., conjugation at the γ-carboxylic functionality of the glutamate moiety of folate) are efficiently recognized by FR in leukemic L1210 cells as well as solid tumor cells (KB and M109 cells).50 In addition, the size of the administered siRNA F

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Figure 4. Treatments of tumor cells with TLPs containing EG5-targeted siRNA (siEG5) or control siRNA (siCtrl) at N/P 16. (a) Metabolic activity of FR-positive KB and FR-negative MCF-7 cells at 48 h after treatment with TLPs. (b) KB cells treated with TLPs observed by DAPI staining under the fluorescent microscope. The mitotic blockade that was induced by EG5 knockdown caused “aster formation” of nuclear DNA (yellow arrows). The scale bars represent 10 μm. (c) mRNA level of EG5 gene measured by qRT-PCR at 48 h after transfection of TLPs in FR-positive KB and L1210 cells. GAPDH was used as housekeeping gene.

endolysosomal compartments after cellular uptake, it is crucial to understand whether the cargo can be released from the endosomal entrapments, reach to the cytoplasm and be available for RNA interference (RNAi) machinery for mediating gene silencing. To shed further light on the siRNA release from TLPs by immuno-TEM, we incubated KB cells with TLP1 (N/P 16) for 4 h (Figure 3a−d). Generally, these TLP1-containing vesicles were intact and surrounded by a continuous membrane. Occasionally, some of these vesicles had a discontinuous membrane, and the electron density of the vesicle content was undistinguishable from that of cytosol (Figure 3a). Moreover, single (Figure 3b) or a small cluster (Figure 3c) of gold particles were spread apparently free in cytosol, often in the close proximity of vesicles with discontinuous membrane. Such escaping siRNA molecules could also be found in the case of other TLP formulations (Figure S9a), and sometimes further away from defective vesicles (Figure S9b). As a key role in the delivery of TLPs, internalized FR proteins (arrowheads in Figure 3b and d) were still associated with siRNA polyplexes. In an attempt to confirm the dislocation of siRNA from endolysosomal compartments,

the results obtained by TEM were complemented by analogous experiments with CLSM. TLP1 was complexed with AF 647labeled siRNA at N/P 16, and KB cells were transfected with CellLight Late Endosomes-RFP and CellLight Lysosomes-GFP. To avoid misleading interpretation based on spatial limitations, 3D reconstruction of serial images from single cell was utilized (Figure 3e and f). As shown in the blue window in Figure 3e, lysosomes (green) were dominant, and late endosomes (blue) were scarcely distributed in the cell. Similarly, siRNA molecules (red) were largely overlapped with late endosomes (purple) or lysosomes (yellow). Notably, although at low frequency, escaping siRNA molecules were detectable (yellow arrowheads in Figure 3e), suggesting TLP1 managed to deliver siRNA into cytosol. One reasonable assumption is that the oligoaminoamides may act as proton sponge motifs to become increasingly cationized during endolysosomal acidification and, supported by the attached fatty acid domains, destabilize the lipid membrane.10,11 Visualizing endosomal release in cells is challenging because very few siRNA release events are detected; for instance, in siRNA LNPs only 1−3.5% of internalized siRNA molecules were delivered into the cytosol.22,38 G

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Figure 5. Endosomal escape capability and siRNA stability of TLPs at N/P 16. (a) Colocalization of siRNA polyplexes with late endosomes and lysosomes was presented by CLSM. Intracellular distribution of AF 647-labeled siRNA (red) polyplexes in KB cells at 2 and 4 h after transfection. Late endosomes and lysosomes were stained with LysoTracker Green (green). Nuclei were stained with DAPI (blue). (b) After transfection for 45 min, colocalization ratios of AF 647-labeled siRNA with late endosomes and lysosomes were calculated using ZEN 2.1 software at 2 and 4 h. (c) Intracellular fluorescence resonance energy transfer (FRET) signals were analyzed by flow cytometry at 1, 4, and 24 h after Cy5/TAMRA doublelabeled siRNA transfection for 45 min in KB and L1210 cells. The samples were excited with a 488 nm laser, and their emission was monitored by using a 660/20 filter. (d) FRET imaging of Cy5 (red)/TAMRA (green) double-labeled siRNA at 1 and 24 h after transfection for 45 min in KB cells by CLSM. Colocalization of the two fluorophores (yellow) indicated intact siRNA. Lysosomes were biolabeled with Lamp1-GFP (artificial color in blue). The scale bars represent 10 μm.

73−88% of luciferase activity was downregulated, which is superior to the parent 356 polyplexes (55%). Among these TLP formulations, most efficient gene silencing activity was achieved by tyrosine-modified TLP1 (88%). At the same time, polyplexes with siCtrl did not induce any unspecific gene silencing. Furthermore, plain cationic 454 polyplexes mediated very modest (