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Fluorinated Oligoethylenimines Nano-Assemblies for Efficient siRNA-Mediated Gene Silencing in SerumContaining Media by Effective Endosomal Escape Tingbin Zhang, Yuanyu Huang, Xiaowei Ma, Ningqiang Gong, Xiaoli Liu, Lu Liu, Xiaoxia Ye, Bo Hu, Chunhui Li, Jianhua Tian, Andrea Magrini, Jinchao Zhang, Weisheng Guo, Jinfeng Xing, Massimo Bottini, and Xing-Jie Liang Nano Lett., Just Accepted Manuscript • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 21, 2018
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Fluorinated
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Oligoethylenimines
Nano-Assemblies
for
Efficient
siRNA-Mediated Gene Silencing in Serum-Containing Media by Effective Endosomal Escape Tingbin Zhang,†,§,‡ Yuanyu Huang,¶,‡ Xiaowei Ma,§,‡ Ningqiang Gong,§ Xiaoli Liu,§ Lu Liu,§,# Xiaoxia Ye,§ Bo Hu,¶ Chunhui Li,¶ Jian-Hua Tian,† Andrea Magrini,ζ Jinchao Zhang,⊥ Weisheng Guo,*, & Jin-Feng Xing,*,† Massimo Bottini§,# and Xing-Jie Liang*,§ †
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, P. R. China
§
Chinese Academy of Sciences (CAS) Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190, P. R. China &
Translational Medicine Center, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, P. R. China. ¶
Advanced Research Institute of Multidisciplinary Science, School of Life Science, Beijing Institute of Technology, Beijing, 100081, P. R. China ζ
Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy. #
Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy. ⊥
Chemical Biology Key Laboratory of Hebei Province, College of Chemistry & Environmental Science, Hebei University, Baoding, 071002, P. R. China
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Abstract Efficient small interfering RNA (siRNA) delivery in presence of serum is of crucial importance for effective gene therapy. Fluorinated vectors are considered to be attractive candidates for siRNA-mediated gene therapy because of their delivery efficacy in serum-containing media. However, the mechanisms driving the superior gene transfection behavior of fluorinated vectors are still not well understood and comprehensive investigations are warranted. Herein, we fabricated a library of perfluorooctanoyl fluoride (PFF)-fluorinated oligoethylenimines (fxOEIs, x is the PFF:OEI feeding ratio), which can readily form nano-assemblies (fxOEI NAs) capable of efficient siRNA delivery in cells cultured in medium both devoid of and supplemented with fetal bovine serum (FBS). The gene silencing test in serum-containing medium revealed that the f0.7OEI/siRNA NAs achieved a luciferase silencing of ~ 88.4% in Luc-HeLa cells cultured in FBS-containing medium, which was almost 2-fold greater than the silencing efficacy of siRNA delivered by the commercially available vector Lipo 2000 (~ 48.8%). High levels of apolipoprotein B silencing were also achieved by f0.7OEI/siRNA NAs in vivo. To assess the underlying mechanisms of the efficacy of gene silencing of fluorinated vectors, two alkylated OEIs, aOEI-C8 and aOEI-C12, were fabricated as controls with similar molecular structure and hydrophobicity to that of f0.7OEI, respectively. In vitro investigations showed that the superior gene delivery exhibited by f0.7OEI NAs derived from the potent endosomal disruption capability of fluorinated vectors in presence of serum, which was essentially attributed to the serum proteins adsorption resistance of the f0.7OEI NAs. Therefore, this work provides an innovative approach to siRNA delivery as well as insights into fluorine-associated serum resistance. Keywords: Fluorine, oligoethylenimine, siRNA delivery, nanostructures, serum resistance, endosomal escape
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Introduction Small interfering RNA (siRNA)-mediated gene therapy is considered to be a promising therapeutic methodology for various congenital and malignant diseases.1-3 Two main classes of gene vectors, namely recombinant viral-based and synthetic vectors, have been developed to improve the delivery of siRNA to specific tissues by protecting the cargo from biological barriers (e.g., nuclease digestion and fast excretion).4, 5 Although recombinant viral-based vectors are efficacious, their translation into clinics is largely hampered by potential immunogenicity and carcinogenicity and by difficulties associated with large-scale fabrication.6-8 Alternatively, synthetic vectors, including cationic polymers9, 10 and liposomes,11, 12 have become popular siRNA delivery agents in virtue of their low immunogenicity, ease of synthesis and structural modification.13 However, most synthetic vectors display a decreased delivery efficacy once exposed to biofluids containing proteins.14-16 This decline is widely attributed to the aggregation of the vectors in presence of proteins.17-19 Therefore, there is an urgent need to develop synthetic vectors that can retain their favorable delivery efficacy in biofluids. Perfluorocarbon (PFC)-based gene vectors have emerged as attractive agents for siRNA-mediated gene therapy during the last few years in virtue of their ability to maintain their delivery efficacy in serum-containing media.20-23 In a pioneering work, Cheng et al. described a class of versatile PFC-modified dendrimers, which maintained their high plasmid DNA (pDNA) delivery efficacy upon addition of 50% (v/v) fetal bovine serum (FBS) to the cell culture medium.24, 25 More recently, You et al. have reported a novel class of bioreducible cationic nanomicelles with a PFC core and exhibiting excellent pDNA delivery efficacy in cells cultured in a medium supplemented with 50% (v/v) FBS.26 The remarkable delivery efficacy of these gene vectors in presence of serum proteins
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was attributed to the vectors’ structural stability as well as steric hindrance against aggregation arising from the poor adsorption of serum proteins onto their surface. However, a comprehensive investigation of the fluorine-mediated structural stability and resistance against serum protein adsorption of PFC-based gene vectors is still absent. In light of the high potential of fluorinated vectors in gene therapy, better understandings of their behavior in presence of serum proteins are warranted. To address these issues, we developed a small library of fluorinated oligoethylenimines (fOEIs) by one-step reaction between perfluorooctanoyl fluoride (PFF) and OEI (Mn = 600 Da). Compared with polyethyleneimine (PEI),27 the “gold standard” of polycation vectors, OEI displays higher biocompatibility but suffers from poor delivery efficacy of genetic materials.28-30 PFF-fluorinated OEIs (fxOEIs, x is the PFF:OEI feeding ratio) were amphiphilic and readily formed liposome-like nano-assemblies (NAs) with the PFF segments densely embedded between the OEI bilayers (Figure 1a). siRNA-loaded f0.7OEI NAs (f0.7OEI/siRNA NAs) achieved a silencing efficacy up to ~ 88.4% even in the presence of serum proteins, which was almost 2-fold greater than the silencing obtained when the commercially available vector Lipo 2000 was used as delivery agent (~ 48.8%). Efficient apolipoprotein B silencing was also achieved by means of f0.7OEI/siRNA NAs in vivo. We also fabricated two types of alkylated OEIs with molecular structure and hydrophobicity, respectively, similar to that of f0.7OEI and used as controls to assess the underlying mechanisms of the efficacy of gene silencing of fluorinated vectors. In vitro investigations showed that the high gene silencing exhibited by f0.7OEI/siRNA NAs in serum-containing medium was due to their efficient cellular internalization and well-maintained ability to escape from the endosomal compartments, which derived from the serum proteins adsorption resistance of the f0.7OEI NAs.
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Results and Discussion
Synthesis and characterization of fluorinated oligoethylenimines Fluorinated oligoethylenimines were obtained by a one-step reaction between PFF and OEI mixed at different ratios (fxOEIs, x is the PFF:OEI feeding ratio) [Figure S1 in the Supporting Information (SI) section]. A small library of fxOEIs was synthesized, including f0.5OEI, f0.7OEI, f1OEI and f1.3OEI. Details of the synthesis and characterization of the fxOEIs are described in the SI. The molecular weights and substitution degrees of the synthetized fxOEIs were characterized by mass spectrometry and ninhydrin assay, respectively (Figures S2 and S3). All the synthetized fxOEIs readily formed spherical nano-assemblies (NAs) via a thin film method. The hydrodynamic diameters (HDs) of the fxOEI NAs were in the range of 128.6-150.6 nm as determined by dynamic light scattering (DLS) (Figure S4). Transmission electron microscopy (TEM) showed that the f xOEI NAs had a liposome-like morphology (Figure 1b), with a membrane most likely formed by the PFF moieties projecting to the inner of a bilayer of OEIs (Figure 1a).31 The critical micelle concentration (CMC) values of the fxOEI NAs were calculated by the Nile red assay and were smaller than 0.080 mg/mL for all the NAs, suggesting their good structural stability (Figure S5).32 The fxOEI NAs were mixed with siRNA to form fxOEI NA-based nano-complexes (fxOEI/siRNA NAs). Agarose gel electrophoresis revealed that all the synthesized fxOEI NAs efficiently retarded migration of the siRNA when the fxOEI NAs:siRNA weight ratio was greater than 5:1 (Figure 1c). As opposed to the fxOEI NAs which were spherical in shape, the fxOEI/siRNA NAs exhibited a multilayer morphology (Figure 1b). This phenomenon was hypothesized due to the strong electrostatic interaction between fxOEI NAs and siRNA, which led to the reassembly of the NAs.
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The cytotoxicity of fxOEI NAs was tested in Luc-HeLa cells by MTT assay in comparison to polyethyleneimine (PEI, Mw = 25 kDa), a polycationic polymer commonly used to condense and transfect siRNA (Figure S6). We found that the toxicity of fxOEI NAs decreased with the degree of PFF substitution decreasing. Additionally, we found that fxOEI NAs showed less cytotoxicity overall than PEI and, when loaded with siRNA, showed little toxicity even at a f xOEI NAs:siRNA weight ratio of 20:1, while most of the cells were killed when incubated with PEI/siRNA complexes at weight ratios greater than 5:1.
Figure 1. Synthesis and characterization of fxOEI NAs. (a) Schematic illustration of the steps to synthesize fxOEIs, fxOEI NAs and the complexes of fxOEI NAs and siRNA. (b) TEM images showing the morphology of the fxOEI NAs and the complexes of fxOEI NAs and siRNA. (c)
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Condensation of siRNA by fxOEI NAs at different vector:siRNA weight ratios characterized by agarose gel electrophoresis (Wvector/WsiRNA = 0 stands for naked siRNA).
In vitro siRNA delivery by fxOEI NAs The intra-cellular delivery of siRNA by fxOEIs was tested by luciferase assay and by fluorescence imaging, flow cytometry and western blotting analyses. The delivery of an anti-luciferase siRNA (anti-Luc siRNA, sequence in Table S1) by fxOEI NAs was evaluated in cells cultured in a medium devoid of or supplemented with 10% (v/v) FBS. The assay was carried out in Luc-HeLa cells, which stably express luciferase. OEI, PEI, and Lipo 2000 were selected as control vectors, whereas a non-targeting siRNA (scrambled siRNA, sequence in Table S1) was used as control gene. As shown in Figure 2a, incubation of Luc-HeLa cells with OEI/siRNA complexes for 48 h had little effect on luciferase expression, indicating the poor gene delivery by OEI. High gene silencing was achieved by PEI/siRNA complexes at a PEI:siRNA weight ratio of 20:1 in cells cultured in FBS-free culture medium. However, the siRNA delivery by PEI was significantly affected by the presence of FBS. Lipo 2000/siRNA complexes achieved a silencing of ~ 62% in cells cultured in absence of FBS, which decreased to ~ 49% in the presence of FBS. In contrast, f0.7OEI/siRNA NAs achieved a silencing of ~ 95% at a NAs:siRNA weight ratio of 20:1 without FBS, which equaled the excellent silencing efficacy of PEI/siRNA complexes. Notably, the siRNA delivery by f0.7OEI NAs was only slightly affected by the presence of FBS (~ 89%). In addition, the gene silencing efficacy of f0.7OEI/siRNA NAs was also challenged in circumstance supplemented with 0%, 10%, 30% and 50% (v/v) FBS, respectively. We found that f0.7OEI/siRNA NAs preserved a potent gene silencing (~ 75%) even in Luc-HeLa cells cultured in a medium containing 50% (v/v) FBS (Figure S7), which validated the favorable gene delivery potentials of these NAs for in vivo applications.
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To further validate the superior siRNA delivery by f0.7OEI NAs, the intra-cellular accumulation profiles of anti-GFP siRNA and anti-PLK1 siRNA mediated by f0.7OEI NAs were assessed (sequences in Table S1). The experiments with anti-GFP siRNA were carried out on GFP-HeLa cells by assessing the GFP silencing via fluorescence imaging (Figure 2b, upper panel). The GFP fluorescence of cells treated with OEI/siRNA complexes was as strong as that of untreated (control) cells, both in absence and presence of FBS, which suggested the low siRNA delivery by OEI. Incubation of cells with Lipo 2000/siRNA complexes and f0.7OEI/siRNA NAs led to a strong decrease in GFP fluorescence in absence of FBS. Almost no fluorescence was detected from cells treated with f0.7OEI/siRNA NAs also in presence of FBS, in agreement with the luciferase assay results. The GFP silencing efficacy in the different treatment groups was also quantified by flow cytometry and was consistent with the results obtained by fluorescence imaging (Figure 2b, bottom panel). OEI/siRNA complexes showed a very low silencing efficacy, while Lipo 2000/siRNA complexes and f0.7OEI/siRNA NAs displayed higher silencing efficacies with or without FBS. The percentage of cells displaying high GFP fluorescence was 60.1% for control (untreated) cells and decreased to 4.18% and 6.17% for cells treated with f0.7OEI/siRNA NAs in the absence and presence of FBS, respectively. The same trend was also obtained for the delivery efficacy of anti-PLK1 siRNA, which was assessed by western blotting. The use f0.7OEI NAs as delivery agent for anti-PLK1 siRNA led to higher knockdown of PLK1 in both the presence and absence of FBS compared to Lipo 2000 (Figure 2c). Taken together, the data obtained from these experiments validated the favorable gene delivery by f0.7OEI NAs also in presence of serum proteins.
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Figure 2. In vitro siRNA transfection by fxOEI NAs. (a) Gene silencing assay in human cervical cancer Luc-HeLa cells with stable luciferase expression. Cells were treated with fxOEI NAs carrying anti-Luc siRNA at different vector:siRNA weight ratios in the absence and presence of 10% (v/v) FBS. PEI and Lipo 2000 served as positive controls. OEI and f0.7OEI/scrambled negative control siRNA (NC siRNA) served as negative controls. Comparison of the delivery of siRNA against (b) GFP and (c) PLK1 in HeLa cells in the absence and presence of 10% (v/v) FBS by OEI, Lipo 2000 and f0.7OEI NAs. The delivery of anti-GFP siRNA was evaluated by measuring the expression of GFP by fluorescence microscopy (upper panels) and flow cytometry (lower panels). The nuclei were stained with Hoechst 33342 (blue) and the GFP was shown in green. Scale bar, 50 μm. The delivery of anti-PLK1 siRNA was evaluated by measuring the PLK1 expression by western blotting. The histograms in (c) represent the quantitative analyses of the PLK1 band intensity for each treatment
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group, normalized to control (untreated cells) by Image J software.
Mechanisms underlying the resistance against serum proteins of fluorinated oligoethylenimines After uncovering the superior siRNA delivery of f0.7OEI NAs in cells cultured in presence of FBS, we set out to explore the mechanisms underlying this phenomenon. It has been widely demonstrated that the molecular structure of hydrophobic segments and the enhanced structural stability they confer are crucial factors contributing to the gene delivery by synthetic vectors.26, 33-35 Thus, for more comprehensive investigations, we synthesized two hydrocarbon (HC)-modified OEIs, named aOEI-C8 and aOEI-C12, by grafting OEI with octanoyl chloride and dodecanoyl chloride, respectively (Figure 3a). The synthesis, purification and characterization of aOEI-C8 and aOEI-C12 are shown in Figures S8-S9. With the length of the alkyl chains equal to that of the PFF chains in f0.7OEI, aOEI-C8 possesses a molecular structure similar to that of f0.7OEI except for the absence of fluorine atoms (Figure 3a). The hydrophobicity of the alkyl chains in aOEI-C12 was similar to that of the PFF chains in f0.7OEI.36, 37 The CMC and zeta potential values of aOEI-C12 NAs and f0.7OEI NAs were almost equal (Figure 3c), implying that they have comparable structural stability. TEM imaging showed that both aOEI-C8 and aOEI-C12 readily formed NAs (Figure 3b). The cytotoxicity of aOEI-C8-, aOEI-C12-, and f0.7OEI-based NAs/siRNA nanocomplexes at different concentrations was tested. Similar concentrations of the nanocomplexes did not elicit significantly different effects on cell viability (Figure 3d). The siRNA delivery by aOEI-C8 NAs, aOEI-C12 NAs, and f0.7OEI NAs was systemically investigated in Luc-HeLa cells cultured in absence and presence of 10% (v/v) FBS by means of the luciferase assay. aOEI-C8/siRNA NAs and aOEI-C12/siRNA NAs achieved a gene silencing of ~ 46% and ~ 73%, respectively, in cells cultured in the absence of FBS (Figure 3e). However, when the cells were cultured in FBS-containing medium, the gene silencing
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achieved by aOEI-C8 NAs and aOEI-C12 NAs significantly decreased to ~ 1.3% and ~ 20%, respectively. This result was explained with a poor resistance against serum proteins of these NAs. Conversely, f0.7OEI NAs achieved a gene silencing of ~ 95% and ~ 85% in cells cultured in medium devoid of FBS and supplemented with FBS, respectively, most likely due to the resistance against serum proteins of f0.7OEI NAs. Taken together, these results suggest that neither the grafted molecular structure nor the enhanced nanostructural stability could account for the resistance against serum proteins of f0.7OEI NAs. Therefore, we hypothesized that this property of f0.7OEI NAs was due to the presence of fluorine atoms, which hamper the adsorption of serum proteins onto the NAs’ surface. Considering the similarity in structure and stability of aOEI-C12 NAs and f0.7OEI NAs, aOEI-C12 NAs were selected for the subsequent studies aimed at validating this hypothesis.
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Figure 3. Physicochemical properties and gene silencing efficacy of alkylated and fluorinated NAs. (a) Chemical structure of aOEI-C8, aOEI-C12 and f0.7OEI. (b) TEM images showing the morphology of aOEI-C8 NAs, aOEI-C12 NAs and f0.7OEI NAs devoid of and loaded with siRNA. Scale bar, 200 nm. (c) CMC and zeta potential values for aOEI-C8 NAs, aOEI-C12 NAs and f0.7OEI NAs. (d) The cytotoxicity of the f0.7OEI-, aOEI-C8- and aOEI-C12- based NAs/siRNA nanocomplexes with various concentrations of NAs determined by the MTT assay. Each treatment was performed in quintuplicate adding 0.2 μg of siRNA in each well. (e) Efficacy of aOEI-C8 NAs, aOEI-C12 NAs and f0.7OEI NAs to deliver siRNA against luciferase in Luc-HeLa cells cultured in medium devoid of and supplemented with 10% (v/v) FBS. ***P < 0.001.
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Because of the differences in gene silencing achieved by aOEI-C12 NAs and f0.7OEI NAs in the presence of serum, the influence of the serum on the cellular uptake and endosomal escape of these vectors was assessed. The aOEI-C12 NAs and f0.7OEI NAs were loaded with Cy5-labelled siRNA (Cy5-siRNA, sequence in Table S1) and their cellular uptake was assessed by flow cytometry and confocal laser scanning microscopy (CLSM). As shown in Figure 4a and Figure 4b, flow cytometry data suggested that f0.7OEI/siRNA NAs accumulated into cells cultured in medium devoid of FBS more efficiently than aOEI-C12/siRNA NAs, which was hypothesized due to the limited miscibility between perfluorocarbon and phospholipids. This property minimizes the fusion of f0.7OEI/siRNA NAs with cell membranes during endocytosis, and improves their cellular uptake.38-40 Addition of FBS to the cell culture medium did not significantly change the efficacy of cell uptake of fluorinated NAs, whereas it led to a significant increase in the uptake of alkylated NAs, albeit these vectors accumulated into cells less efficiently than f0.7OEI NAs also in presence of FBS. The CLSM data were consistent with the flow cytometry data (Figure S10). Following cellular uptake, vectors are usually trapped into the endosomal compartments. This phenomenon represents an obstacle to the vector-mediated siRNA delivery and the endosomal escape of nucleic acid into cytoplasm is crucial for efficient gene silencing.41, 42 In order to assess the endosomal escape of siRNA delivered by fluorinated and alkylated vectors, the intra-cellular trafficking profile of Cy5-labelled siRNA loaded into aOEI-C12 NAs and f0.7OEI NAs was assessed in Luc-HeLa cells stained with Lysotracker Green via CLSM (Figure 4c). The colocalization ratio (CR) between siRNA and lysosomal compartments was calculated by Image-Pro Plus 6.0 software and used as a negative index of the endosomal escape efficacy of the NAs/siRNA complexes (Figure 4d). CLSM images showed a similar number of diffused cytoplasmic red spots in cells treated with
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both f0.7OEI NAs and aOEI-C12 NAs in absence of FBS. This result suggested that both the NAs efficiently disrupted the endosomes, most likely because of their weak base nature. However, upon addition of FBS to the cell culture medium, the red spots became colocalized with the green ones in cells treated with aOEI-C12 NAs, but not in those treated with f0.7OEI-NAs (Figure 4c). CR data validated the CLSM-based results. The siRNA delivered by f0.7OEI NAs in absence of FBS had a CR of ~ 48% that was smaller than the CR of siRNA delivered by aOEI-C12 NAs (~ 57%) (Figure 4d). Addition of FBS to the culture medium led to a non-significant increase in the CR value of the siRNA delivered by f0.7OEI NAs (~ 54%), whereas it led to a strong and significant increase in the CR value for the siRNA delivered by aOEI-C12 NAs (~ 81%) (Figure 4d). These data suggest that the serum proteins markedly affected the endosomal escape capability of aOEI-C12 NAs, but not that of f0.7OEI NAs. The effects of serum proteins on the endosomal escape capability of siRNA delivered by aOEI-C12 NAs and f0.7OEI NAs were confirmed via luciferase assay on Luc-HeLa cells cultured in absence and presence of 10% (v/v) FBS (Figure 4e). For a more comprehensive evaluation on the effects of serum proteins, chloroquine and bafilomycin A1 were introduced during the NA-mediated gene delivery to stimulate and inhibit, respectively, the endosome disruption. Chloroquine is a weak base that promotes endosome disruption and subsequent cargo escapes from endosomes,43 whereas bafilomycin A1 prevents the acidification of endosomes thus inhibiting the effects of weak bases on the stability of endosomes.44 As shown in Figure 4e, the gene silencing achieved by aOEI-C12/siRNA NAs was significantly improved from ~ 20% to ~ 40% in serum-containing medium in the presence of chloroquine, whereas the effect of bafilomycin A1 decreased, albeit not-significantly, the siRNA delivery by aOEI-C12 NAs. Conversely, the gene silencing achieved by f0.7OEI/siRNA NAs was significantly compromised by bafilomycin A1,
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irrespective of the presence of the serum in the culture medium, validating the conclusion that the superior siRNA delivery by f0.7OEI NAs was due to the ability of fluorinated vectors to induce the disruption of the endosomal compartments even in presence of serum proteins. These conclusions were also validated by means of CLSM. As shown in Figure 4f and Figures S11-S12, the addition of chloroquine led to an increase in the number of cytoplasmic diffused red spots in cells incubated with aOEI-C12/siRNA NAs, whereas addition of bafilomycin A1 led to the disappearance of the red spots in cells treated with f0.7OEI/siRNA NAs. Collectively, these data showed that the hampered gene silencing efficiency of siRNA delivered by aOEI-C12 NAs in presence of FBS stems from the decreased endosomal escape ability of alkylated vectors upon the addition of serum despite the 2-fold increase in cellular uptake. Conversely, the gene silencing efficiency of siRNA delivered by f0.7OEI NAs stemmed from the property of fluorinated vectors to display high cellular uptake efficacy and well-maintained endosomal disruption capability in presence of serum.
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Figure 4. Influence of serum proteins on the cellular uptake and endosomal escape of siRNA delivered by alkylated and fluorinated NAs. (a) Cellular uptake of siRNA delivered by aOEI-C12 NAs and f0.7OEI NAs quantified by flow cytometry 4 hours after incubation. (b) Mean fluorescence intensity determined by flow cytometry for the different cell treatment groups. (c) Intracellular distributions of siRNA delivered by aOEI-C12 NAs and f0.7OEI NAs imaged by confocal laser scanning microscopy (CLSM) 8 hours after incubation. siRNA was labeled with Cy5 (red) and
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lysosomes were stained with Lysotracker Green (green). Scale bars are 20 μm. (d) Co-localization ratio (CR) of Cy5-labelled siRNA and Lysotracker Green calculated using Image-Pro Plus 6.0 software. (e) Influence of chloroquine and bafilomycin A1 on the luciferase silencing efficacy of siRNA delivered by aOEI-C12 NAs and f0.7OEI NAs in Luc-HeLa cells cultured both in the absence and presence of 10% (v/v) FBS. (f) CLSM images show the intracellular distributions of f0.7OEI/siRNA and aOEI-C12/siRNA 8 hours after incubation. The concentrations of chloroquine and bafilomycin A1 were 50 μM and 200 nM, respectively. Scale bars are 20 μm. ns = not significant (P > 0.05), **P < 0.01, ***P < 0.001.
Next, we investigated if the observed differences in the in vitro gene delivery by aOEI-C12 NAs and f0.7OEI NAs in the presence of serum proteins was due to the degree of resistance of these NAs against the adsorption of serum proteins onto their surface. To this end, the NAs were incubated in FBS-containing medium, collected by centrifugation and the amount of serum proteins adsorbed onto their surface was measured by bicinchoninic acid (BCA) assay and polyacrylamide gel electrophoresis (PAGE). Although aOEI-C12 NAs and f0.7OEI NAs had nearly equal surface charge, the amount of serum proteins adsorbed onto f0.7OEI NAs was much lower than that adsorbed onto aOEI-C12 NAs, as quantified by BCA assay (Figure 5a) and PAGE (Figure 5b). The affinity between serum proteins and fluorinated and alkylated NAs was further evaluated by quartz crystal microbalance (QCM). Bovine serum albumin (BSA), the most abundant proteins in plasma, was chosen as model protein45. As shown in Figure 5c and Figure 5d, the changes in frequency – due to the adsorption of NAs onto the albumin-coated electrode – were greater for aOEI-C12 NAs with respect to f0.7OEI NAs for all the NA concentrations tested, indicating that BSA had a greater affinity for the surface of aOEI-C12 NAs relatively to the surface of f0.7OEI NAs. The influence of serum proteins on the colloidal stability of the NAs was also studied. aOEI-C12 NAs were stable in
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FBS-free medium for 2 hours, however they exhibited poor colloidal stability in FBS-supplemented medium as suggested by the increase in HD (Figure 5e) and solution turbidity (Figure 5f) with time. By contrast, the HD of f0.7OEI NAs was not affected by the presence of FBS, validating the colloidal stability of these NAs due to the resistance against the adsorption of serum proteins onto their surface. These results suggested that the observed differences in the in vitro gene delivery by aOEI-C12 NAs and f0.7OEI NAs in the presence of serum proteins was due to the different degree of adsorption of serum proteins onto their surface. In particular, the distinctive characteristic of protein adsorption resistance endowed f0.7OEI NAs with high siRNA delivery efficacy even in presence of serum proteins (Figure 5g).
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Figure 5. Influence of serum proteins on the stability of alkylated and fluorinated NAs. The amount of proteins adsorbed onto aOEI-C12 NAs and f0.7OEI NAs was quantified by (a) BCA assay and (b) polyacrylamide gel electrophoresis. mp, amount of protein; mv, amount of vector. ***P
