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. 2020 Feb 11;6(2):174–188. doi: 10.1021/acscentsci.9b01052

Virus-like Nonvirus Cationic Liposome for Efficient Gene Delivery via Endoplasmic Reticulum Pathway

Xiaoling Yuan 1, Bing Qin 1, Hang Yin 1, Yingying Shi 1, Mengshi Jiang 1, Lihua Luo 1, Zhenyu Luo 1, Junlei Zhang 1, Xiang Li 1, Chunqi Zhu 1, Yongzhong Du 1, Jian You 1,*
PMCID: PMC7047280  PMID: 32123735

Abstract

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Gene vectors play a critical role in gene therapy. To achieve efficient transfection, we developed a novel nonvirus cationic liposome (Lipo-Par), which was bound covalently with the cationic polypeptide pardaxin (Par). Interestingly, the Lipo-Pars exhibited highly enhanced gene transfection efficiency in various cell lines compared to that of the non-Par-bonded liposomes (Lipo-Nons). As a result, the internalization and intracellular transport mechanisms of the Lipo-Pars were investigated, and the findings indicated their ability to actively target the endoplasmic reticulum (ER) by moving along the cell cytoskeleton after undergoing caveolin-mediated endocytosis. This intracellular transport process is similar to that of some viruses. It was also found that ER stress and calcium level disturbances can affect the Lipo-Par-mediated expression of certain exogenous genes. A possible, yet non-negligible explanation for the high transfection efficiency of the Lipo-Par is its virus-like intracellular behavior and the intimate relationship between the ER membrane and the nuclear envelope.

Short abstract

To take advantage of the high efficiency of virus vectors and make up for its shortcomings, a virus-like nonvirus cationic liposome was designed, which can target ER and achieve exciting efficiency.

Introduction

The past half-century has witnessed overwhelming momentum and remarkable breakthroughs in gene therapy, which corrects genetic abnormalities or compensates for genetic defects, distinguishing itself in areas such as neuromuscular diseases, hemophilia, immunodeficiency, and cancer. Acting as a link between therapeutic genes and regulatory expression systems, gene delivery systems play a key role in gene therapy. Therapeutic genes and regulatory gene expression systems can be unified only when the safety and efficiency of the gene delivery systems are ensured, an outcome that is important in overcoming inefficiency and preventing serious side effects in treatment. Currently, vectors used for gene therapy mainly fall into three categories: virus, virus-like, and nonvirus carriers. In terms of clinical cases, adenoviral, retroviral lentiviral, and other viral vectors account for 20.5% (n = 547), 17.9% (n = 478), and 7.3% (n = 196) by 2017, respectively, which is 4.66-fold that of liposomes (4.4%, n = 117).1 Obviously, viral vectors constitute a large share in the field of gene therapy, which is mainly because of their efficient transfection rate. Despite its extremely simple composition and structure, the virus has practiced the “obscuration method” over millions of years of evolution, evading immune recognition systems and disguising itself as “an insider”. With the help of the delicate cellular structure of the host cell and its signal transduction pathways, the virus craftily realizes its own transport and infection. The virus infects the host cell, primarily in interphase, using three main steps—binding to cell surface receptors or adhesion factors, entering through endocytosis, and inoculating a replication-active genome into the cell nucleus. For viruses, there are four challenges to overcome to achieve successful infection. First, to solve the problem of internalization, the virus needs to bind to multiple specific adhesion factors or receptors on the cell surface, thereby reducing the possibility of being off target. Second, the virus needs to adjust its pH carefully to shed its coating and penetrate in the weak acidic environment of the early and late endosomes, thereby avoiding being transported to lysosomes.2,3 Third, due to the crowded intracellular environment, the virus needs to disguise itself as an endogenous cargo, and as soon as it hijacks dynein, it can move along the microtubules and approach the cell nucleus. Fourth, to localize to a nuclear pore site, the viral surface proteins must bind to the nuclear import receptors (e.g., importin 7, importin α, importin β) presented on the nuclear pores.4,5 The size of the material allowed to pass through the nuclear pore is strictly limited (less than 39 nm). Therefore, after being localized to the nuclear pore, a small virus or capsid, such as the parvovirus (18–24 nm) and hepatitis B virus (36 nm), can enter the nucleus with its original structure intact, but a virus larger than the nuclear pore must go through deformation or depolymerization to introduce their active genome into the nucleus.

Simian virus (SV40) enjoys a unique means of internalization and intracellular transport and is able to infect host cells efficiently. For internalization, SV40 binds to the GM-1 ganglioside on the surface of CV-1 cells and then moves along the cell membrane to find glycosphingolipids or cholesterol- or caveolin-1-enriched microdomains, where it binds to caveolin-1 and invaginates into a flask-shaped caveola.6 It is well-known that caveolin-mediated endocytosis is correlated to a lesser extent with lysosomal degradation pathways than clathrin-mediated endocytosis. In addition, SV40 activates the tyrosine kinase-induced signal cascade to induce local microfilaments depolymerization, thereby breaking the cortical cytoskeleton barrier that can block the entrance of a foreign substance.7 Then, SV40 moves along the microtubules until it directly reaches the endoplasmic reticulum (ER) around the nucleus, where it sheds its coating completely, and the genome is released near the nuclear pore.

Despite the undeniable advantages of transfection efficiency, viral vectors (such as lentivirus, adenovirus, and retroviral vector) are still controversial in clinical applications because of the gene mutations and immunogenicity. In recent years, virus-like and nonviral gene vectors have attracted much attention due to their superior safety and biocompatibility compared to that of viral gene vectors.

Cationic liposomes, a classical nonviral gene vector, make use of their positive charge to electrostatically interact with the negative charge of DNA to form a liposome/DNA complex, which can enter the cell by endocytosis or membrane fusion to achieve internalization and integrate its therapeutic genes. However, the level of their transfection efficiency has been uninspiring and creates a critical bottleneck during the development of cationic liposomes. Similar to the challenges faced by infection-ready viruses during the infection of host cells, nonvirus vectors also need to overcome three challenges to mediate gene transfection: the internalization barrier, lysosomal degradation, and nuclear membrane barriers. Unlike viruses, however, nonviral vectors have enhanced difficulty in effectively overcoming these challenges due to their lack of corresponding biological characteristics. Consequently, most nonviral vectors exhibit poor transfection efficiency, which severely limits their clinical application. The efficient gene delivery mechanism of the viruses represented by SV40 is of great significance for use in the design and preparation of highly efficient nonviral gene vectors. With reference to the intracellular nonlysosomal transport pathway of the SV40 virus, specifically its ability to target the ER and move along the cytoskeleton to reach the nucleus and release its genetic material, we designed an SV40-like nonviral cationic liposome, Lipo-Par. Structurally, the main constituent of the Lipo-Par, phospholipid DSEP-PEG2000-NH2, is covalently bound to the cationic peptide pardaxin by a condensation reaction. Pardaxin, also known as GE33, is a 33-amino-acid cationic antimicrobial peptide derived from the Red Sea Moses sole Pardachirus marmoratus that has hydrophobic and pore-forming properties8−10 and can insert into phospholipid bilayers,11 rapidly localize to the ER through a nonlysosomal intracellular transport pathway, and cause an ER stress response.12 We hypothesized that cationic liposomes modified with pardaxin can utilize cell internalization and intracellular transport mechanisms similar to those of SV40 and similarly localize to the ER. Therefore, this nonviral vector was thought to exhibit significantly enhanced gene delivery performance; that is, it is expected to have significantly improved gene transfection efficiency. To this end, we first investigated the transfection efficiency of the vector that was carrying GFP plasmids into different cancer cells of different lineages and then studied the vector-mediated gene transport process to determine its unique delivery mechanism. It is well-known that the ER membrane is connected to the extranuclear membrane, and the cytoskeleton and the ER have important roles in the composition and reorganization of the nuclear membrane.13−16 Based on this intimate relationship between the ER membrane and the nuclear membrane, delivering exogenous genes to the ER may be an effective way to increase the efficiency of gene transfection.

Experimental Section

Reagents

Commercial transfection reagent Lipofectamine 2000 was purchased from Invitrogen, USA. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from Avanti, USA. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000-NH2) were purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd. Pardaxin polypeptide (GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE) was synthesized by Shanghai Qiang Yao Biotechnology Co., Ltd. The raw material di-tert-butyl dicarbonate ((BOC)2O) used for the synthesis of DSPE-PEG2000-Par was purchased from Aladdin Reagent (Shanghai) Co., Ltd. 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), the materials for the initial synthesis of DSPE-PEG2000-Par, were purchased from Sigma-Aldrich. Company, USA. Fluorescein isothiocyanate (FITC) and DiD perchlorate were purchased from Dalian Meilun Biotechnology Co., Ltd. Nonbiologically functional FITC-DNA (5′-FITC-CAGACCGACTGGATCT-3′) was synthesized by Shanghai Jierui Biological Engineering Co., Ltd. Hoechst 33342, Lyso-Tracker, ER-tracker, a cell cycle and apoptosis analysis kit, and diethyl pyrocarbonate-treated water were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Chlorpromazine (CPZ), filipin, indomethacin, colchicine, amiloride hydrochloride, nocodazole, 2,6-dimethyl-β-cyclodextrin (M-β-CD), brefeldin A (BFA), and calcium chelating agent 1,2-bis (2-aminophenoxy) ethane N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) were purchased from Dalian Meilun Biotechnology Co., Ltd. KIRA6 was purchased from MedChemExpress Co., Ltd., USA. Paclitaxel was purchased from Jiangsu Taxus Pharmaceutical Co., Ltd. Chloroform (CHCl3), dimethyl sulfoxide (DMSO), and formaldehyde (CH2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. Fetal bovine serum (FBS) was purchased from Gibco, USA. Milli-Q water was used in the experiments, and the reagents used were of analytical grade.

Cell Lines

The following cells lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences: HEK 293T cells (human embryonic kidney transformed cells), MCF 10A cells (human normal mammary epithelial cells), MCF-7 cells (human breast cancer cell), MCF-7 (PDX) cells (human breast cancer cell), SKOV3 (human ovarian cancer cell), and HeLa cells (human cervical cancer cells). Live cells were kept at 37 °C in a humidified atmosphere containing 5% CO2 and cultivated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin sulfate. MCF 10A cells were cultivated in DMEM/F12 containing 5% horse serum, 20 ng/mL EGF, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1% NEAA, and 1% P/S.

Synthesis of DSPE-PEG2000-Par (Figure S1)

DSPE-PEG2000-Par was obtained by a condensation reaction between the amino group of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000-NH2) and the carboxyl group of pardaxin polypeptide. First, we prepared an anhydrous DMF solution of pardaxin and added (BOC)2O in DMF to protect the free amino group of the pardaxin polypeptide, and the reaction was carried out for 12 h in the dark. After the protection reaction was completed, the carboxyl groups on the pardaxin polypeptide were activated by adding EDC and NHS to increase the coupling efficiency. After the carboxyl group was activated for 2 h, DSPE-PEG2000-NH2 was added, and the reaction was allowed to continue under magnetic stirring for 24 h. When the condensation reaction was complete, the appropriate amount of HCl was added to remove the BOC protection, the solution was stirred for 2 h, and the pH of the solution was readjusted.

Preparation and Characterization of the Cationic Liposome

A Lipo-Par consists of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000-NH2), and DSPE-PEG2000-Par, which was prepared according to the experimental procedure described above. All materials were dissolved in chloroform and sonicated before use. Briefly, the liposomes were prepared through the thin film dispersion method, and the obtained liposome film was hydrated with DEPC-treated water. A similar method was used to prepare a nonmodified cationic liposome (Lipo-Non) to use as a control. The size and zeta potential of the liposomes were measured by a photodynamic scattering instrument (Malvern, UK), and the morphology and structure were characterized by transmission electron microscopy (JEOL JEM-1230, Japan).

Plasmid and Gene Transfection

An EGFP plasmid (pEGFP) was obtained that encoded only the reporter gene EGFP. mCherry-ER was a gift from Michael Davidson (Addgene plasmid #55041). mEmerald-tubulin-6 was a gift from Michael Davidson (Addgene plasmid #54291). All plasmids were extracted and purified by Sunny Biotech Co. Ltd., Shanghai. For gene transfection, cells were seeded at 5 × 104 cells per well in a 48-well dish ∼16 h before transfection experiments were undertaken. Cationic liposome/pDNA complexes were prepared at different mass ratios [LipofectamineTM 2000: pEGFP = 1.5:1; DOTAP (Lipo-Par): pEGFP = 6:1; and DOTAP (Lipo-Non): pEGFP = 6:1]. The cells were transfected for 2 h without FBS, and the amount of plasmid DNA was 0.5 μg per well. After plasmid transfection, fresh medium containing 10% FBS was added to the cultured cells for an additional 6 or 22 h, and fluorescence microscopy was performed.

Cellular Uptake Experiments

Cells were seeded at 2.5 × 104 cells per well in a 48-well dish ∼16 h prior to the uptake experiments. The cells were pretreated with clathrin-mediated uptake inhibitor (CPZ, 25 μM), caveolin-mediated uptake inhibitors (filipin, 5 μg/mL; indomethacin, 50 μM), macropinocytosis inhibitors (amiloride, 50 μM; colchicine, 25 μM), and lipid raft-mediated uptake inhibitor (M-β-CD, 10 μg/mL) for 30 min. The DiD-labeled Lipo-Pars and Lipo-Nons were diluted to 15 μg/mL with DMEM without FBS and incubated with cells for 2 h. The cells were then washed three times with PBS and fixed, and images of the cells were taken with an inverted fluorescence microscope (AIR, Nikon, Japan). The images were viewed, processed, and analyzed using ImageJ.

High-Resolution Laser Confocal Microscopy

Images of the fixed cells were captured at room temperature. The live cells were kept at 37 °C in a live-cell incubation chamber. Images of the treated-cell imaging was performed with a high-resolution laser confocal microscope (LSM 880 with Airyscan, Carl Zeiss Jena, Germany). The images were taken at 100× with a numerical aperture of 1.4 or at 63× with a numerical aperture of 1.4 and an oil objective (Zeiss). Multichannel photos are captured on the same focal plane. The images were viewed, processed, and analyzed using Imaris 9.3.1 as well as ImageJ.

Quantitative Analysis by Flow Cytometry

The cells were seeded at 2 × 105 per well in a 6-well dish for 16 h before the experiments were performed. pEGFP transfection and propidium iodide (PI) staining was performed according to the protocol described above and the experimental manual of the cell cycle and apoptosis assay kit (Beyotime). The treated cells were digested with 0.25% trypsin and resuspended to 2 × 107/mL with buffer, and then loaded for flow analysis (Cytomic FC 500 MCL, Beckman Coulter, USA).

Quantitative Analysis by Western Blotting

The cells were seeded in a 6-well dish at 2 × 105 per well and cultured for 16 h in DMEM containing 10% FBS, and two replicate wells were used for each experimental group. The cells were treated with DMEM (control), Lipo-Pars (15 μg/mL), and Lipo-Nons (15 μg/mL) for 2 h. The cells were washed twice with precooled PBS buffer and then treated with cell lysate (RIPA:PMSF = 100:1, Biyuntian) on ice for 30 min. The cells were scraped, collected in EP tubes, and centrifuged at 12,000 rpm for 5 min. The protein supernatant was added with loading buffer and boiled to use as an immunoblot sample and stored at 4 °C. After it was loaded, the gel was run under constant pressure until the protein marker (Hangzhou Fude Biological Technology Co., LTD) separated. The proteins were transferred to the NC membrane, which was then blocked, and probed successively with primary and secondary antibodies. The fluorescence was detected with an imager (C428-G-BOX, Syngene, UK) and quantified using ImageJ.

Image Analysis and Statistical Analysis

All data are representative of multiple independent experiments. To conduct the best possible analysis of the results from the cell uptake, gene transfection, and colocalization experiments, all experiments were performed independently at least three times. All presented data are representative, and the representative cells are enlarged. The fluorescent images were viewed, processed, and analyzed by Imaris 9.3.1 and ImageJ. All fluorescent images are presented with corresponding scale bars. The error bars are shown in a chart columns.

Safety Statement

No unexpected or unusually high safety hazards were encountered.

Results

Synthesis and Characterization of the Lipo-Pars

To synthesize ER-targeting cationic liposomes, we first synthesized DSPE-PEG2000-Par by covalently binding the carboxyl group of pardaxin (Figure 1A) to the amino group of DSPE-PEG-NH2 (Figure S1). Then, the Lipo-Pars were prepared by thin film dispersion using DOTAP (cationic phospholipid), DOPE (neutral phospholipid), DSPE-PEG2000-NH2, and DSPE-PEG2000-Par (Figure 1B, right). Similarly, the nontargeting liposome Lipo-Nons were prepared with DOTAP, DOPE, and DSPE-PEG2000 (Figure 1B, left) and used as controls. The sizes of the two liposomes were normally distributed and were approximately 120 nm, and the zeta potential of the Lipo-Pars was approximately 34 mV, while that of the Lipo-Nons is approximately 27 mV (Figure S2A). The two types of liposomes were imaged by transmission electron microscopy, and a full spherical shape and distinct membrane structure were observed (Figure 1C). Additionally, it was shown that Lipo-Nons and Lipo-Pars remained stable in water for days and in DMEM culture media for at least 8 h (Figure S2H, Figure S2I), and the cell viability when cells were cultured with 40 μg/mL Lipo-Par for 24 h was higher than 50% (Figure S2G), which supported further research. The concentration we used in transfection experiments is 12 μg/mL, while liposomes/pEGFP complexes were internalized for 2 h.

Figure 1.

Figure 1

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Synthesis of a novel cationic liposome and its gene transfection efficiency. (A) Three-dimensional schematic of the antimicrobial peptide pardaxin. The pardaxin sequence, NH2-G-F-F-A-L-I-P-K-I-I-S-S-P-L-F-K-T-L-L-S-A-V-G-S-A-L-S-S-G-G-Q-E, is composed of two α-helices with a proline residue acting as a hinge between the two helices. The structural image was acquired from the NCBI protein data bank (https://www.ncbi.nlm.nih.gov/protein/1502235A). (B,C) Schematic and characterization of the Lipo-Par and Lipo-Non cationic liposomes. The scale bar of (C) represents 500 μm. (D,E) pEGFP transfection efficiency of the different cationic liposomes in different cell lines is shown. The mass ratio of the Lipofectamine 2000 (Lipo-2000) to pEGFP was 1.5:1. The mass ratio of the Lipo-Par or Lipo-Non to pEGFP was 6:1. (E) Results from the quantitative analysis of (D) using ImageJ. The scale bar represents 500 μm. (F) Agarose gel electrophoresis results. The mass ratios of the liposomes to pEGFP, from left to right, are 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, and 6:1. When the ratio was 6:1, the DNA was tightly bound to the two liposomes. (G) DiD-lipo and FITC-DNA complexes were synthesized. The sequence of nonbiological functional FITC-DNA is 5′-FITC-CAGACCGACTGGATCT-3′. The mass ratio of the liposomes to the DNA was 6:1. The liposome is green, DNA is red, and the overlapping parts are yellow. The scale bar represents 500 μm. (H,I) FITC-DNA delivery of different cationic liposomes after 24 h treatment is shown. Cells were cultured with liposomes/DNA complexes for 2 h and cultured with fresh media for an additional 22 h. The scale bar of (H) represents 200 μm. (I) Results from the quantitative analysis of (H).

pGFP Transfection Research

It was found that both the Lipo-Pars and Lipo-Nons can form tight complexes with the negatively charged plasmid DNA with the optimal mass ratio of 6:1 (Lipo: plasmid, W/W), and no free DNA was observed (Figure 1F). We coincubated the DiD-labeled liposomes with FITC-labeled ssDNA, and using fluorescence microscopy, we observed that the fluorescence associated with each complex was fully merged, which further confirms that they both formed stable liposome/DNA complexes (Figure 1G). In vitro transfection experiments on different cancer cell lines revealed that the cationic antibacterial peptide pardaxin has a greatly desired effect by promoting gene delivery efficiency. Compared with that mediated by the Lipo-Nons, the Lipo-Par-mediated transfection significantly increased the expression of the exogenous genes. For example, the GFP expression levels in the HEK 293T, MCF-7, MCF-7 (PDX), SKOV3, and HeLa cells were 28-, 58-, 96-, 419-, and 50-fold higher, respectively, when mediated by Lipo-Pars compared to the expression in the Lipo-Nontransfected cells (Figure 1D,E). Moreover, our experiments showed that, although the GFP expression in the MCF-7 cells mediated by Lipo-Pars was not significantly different from that induced by Lipofectamine 2000 (a commonly used commercial transfection reagent, abbreviated herein as Lipo-2000), the Lipo-Pars exhibited better transfection efficiency in the HEK 293T, MCF-7 (PDX), SKOV3, and HeLa cells (Figure 1D,E). Additionally, the transfection efficiency mediated by Lipo-Par in MCF 10A (human normal mammary epithelial cells) was significantly lower than in MCF-7 (Figure S2D). These results demonstrate that the cationic liposomes modified with pardaxin can significantly enhance transfection efficiency in most cancer cells, which may be attributed to the unique impact that the pardaxin exerts on the intracellular fate of these cationic liposomes.

Gene Internalization Experiments

Lipo-Pars, Lipo-Nons, and Lipo-2000s were separately formed complexes with FITC-labeled DNA, and MCF-7 cells were incubated with each. The study found that, compared to those incubated with the Lipo-Nons, the cells incubated with the Lipo-Pars showed a significantly stronger fluorescence signal, indicating that more DNA sequences were completely internalized with increasing incubation time (Figure S2E,F). DNA internalization efficiency mediated by the Lipo-Pars was even higher than that of the Lipo-2000s at 8 and 24 h, and the difference between the two liposomes was much more obvious at 24 h (Figure 1H,I, Figure S2B, S2C), which means that DNA delivered by the Lipo-Pars effluxes to a lesser extent than that delivered by the Lipo-2000s. This finding indicates that the liposomes modified with pardaxin can carry more DNA into the cell and may enhance the cytoplasmic retention of the DNA, which provides more opportunities for exogenous genes to enter the nucleus and be expressed.

Internalization Mechanism of the Lipo-Pars

Liposome uptake is usually an ATP-consuming process and is undertaken by the following mechanisms: clathrin-mediated endocytosis, caveolin-mediated endocytosis, lipid raft-mediated endocytosis, macrocytosis, etc. (Figure 2A). To investigate how Lipo-Pars and Lipo-Nons enter cells, different inhibitors of the endocytic pathways were used, including chlorpromazine (a clathrin-mediated endocytosis inhibitor),17 filipin/indomethacin (inhibitors of caveolin-mediated endocytosis),18−21 methyl-β-cyclodextrin (an inhibitor of lipid raft-mediated endocytosis and used to inhibit caveolin-mediated endocytosis as well),26,27 and amiloride/colchicine (inhibitors of macrocytosis).22−25 The cells were pretreated with each inhibitor for 30 min and then incubated with DiD-labeled Lipo-Pars or Lipo-Nons. The results showed that the lipid raft structure, an essential component of caveosome, had an important effect on the uptake of the Lipo-Pars because, when it was destroyed with M-β-CD (a cholesterol depletor), the uptake of Lipo-Pars was reduced dramatically (Figure S3B). From the microscopic images, the liposomes in this group remained on the surface of the plasma membrane and few entered the cells (Figure 2B). Together with the evidence of reduced internalization of Lipo-Pars in filipin/indomethacin/M-β-CD group and partial colocalization of caveolin-1 and the Cy3-DNA carried by the Lipo-Pars (Figure S3D), it was demonstrated that the Lipo-Pars were mainly internalized through the caveolin-mediated endocytic pathway. Similarly, upon clathrin-mediated endocytosis or macrocytosis inhibition, the number of internalized Lipo-Nons significantly decreased in the cells, indicating that the Lipo-Nons enter cells mainly through these two pathways (Figure 2B, Figure S3B). Theoretically, clathrin-coated vesicles and macropinosomes easily pass through the early endosome-late endosomal-lysosomal pathway (Figure 2A) downstream, rendering the DNA extremely unstable in the lysosomal acidic environment. Studies have shown that caveolin-mediated endocytic vesicles, however, form caveosomes that can escape lysosomal capture,6 which enables the genes to cross the internal organelle barrier and eventually reach the nucleus.

Figure 2.

Figure 2

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Internalization mechanism of the Lipo-Pars. (A) Schematic of Lipo-Par and Lipo-Non internalization. Common internalization pathways include those that are clathrin mediated, caveolin mediated, and macropinocytosis. The novel cationic liposome Lipo-Par enters cells mainly through the caveolin-mediated pathway, which is known to follow a nonlysosomal route. (B) Results from the investigation into Lipo-Par and Lipo-Non internalization. Cells were pretreated for 30 min with different chemical inhibitors to block relevant internalization pathways (CPZ, 25 μM; filipin, 5 μg/mL; indomethacin, 50 μM; amiloride, 50 μM; colchicine, 25 μM; and M-β-CD, 10 μg/mL) and then incubated with DiD-labeled Lipo-Pars or Lipo-Nons for 2 h. Images of representative single cells are enlarged. The lipid raft, a key component of caveosomes and clathrin-coated vesicles, plays a key role in both Lipo-Par and Lipo-Non uptake. Each liposome can enter cells in different ways, but the internalization of the Lipo-Pars was mediated mainly by caveolin and that of the Lipo-Nons was mediated mainly by clathrin. The scale bar represents 100 μm. (C) Results from the quantitative analysis of the flow cytometry experiments (values are from the values of the non-Blank group minus the value of the Blank group (1.1%) in the Figure S2A). Cells were pretreated for 30 min with different chemical inhibitors and transfected with pEGFP for 2 h, cultured for an additional 22 h. The results show that blocking a single uptake pathway generally did not affect the gene delivery efficiency compared to the efficiency of the control group. However, colchicine, a macropinocytosis and microtubule inhibitor, greatly affected the transfection of the Lipo-Pars. The experiment was done once. (D,E) Images show the efflux of DNA carried by the Lipo-Pars and Lipo-Nons. Both Lipo-Par/FITC-DNA (red) and Lipo-Non/FITC-DNA (red) were allowed to internalize for 8 h and were observed in the same field of view at 24 and 48 h. Compared to the Lipo-Nons, the Lipo-Pars carried more DNA into the cells, and the carried DNA stayed in cells longer. (E) Results from the quantitative analysis of (D) using ImageJ. The scale bar represents 200 μm. The experiment was done three times.

To test whether different types of internalization affects the plasmid transfection efficiency, we compared the levels of GFP expression in each group by flow cytometry (Figure 2C, Figure S3C). It was found that, consistent with the microscopic imaging results (Figure 1E,I), the transfection efficiency of the Lipo-Pars was significantly higher than that of the Lipo-Nons in general. Additionally, inhibiting clathrin- (using CPZ) or caveolin- (using filipin/indomethacin) mediated internalization had little effect on the pEGFP transfection efficiencies of Lipo-Nons or Lipo-Pars, respectively. The pretreatment with M-β-CD, a cholesterol depleting agent which was widely used as the inhibitor of caveolin-mediated internalization as well,28,29 however, pulled the transfection efficiency down to almost zero (Figure S3A). The inconsistent transfection results of Lipo-Pars in filipin/indomethacin group and M-β-CD group are confusing, and we speculated that the extreme inefficiency in M-β-CD group might be due to its rather low uptake efficiency. In contrast, there were still Lipo-Pars entering the cells in the filipin/indomethacin groups, which hinted that other unclear effective internalization pathways made a difference in the transfection of Lipo-Pars (Figure 2C, Figure S3A,B). Besides, after using colchicine to inhibit macropinocytosis and tubulin assembly, the transfection efficiency of the Lipo-Pars was greatly reduced, possibly due to the importance of the microtubules in caveosome trafficking. We assume that, when lacking microtubules, as is described for the “high-speed transport channel”, the Lipo-Pars cannot achieve subcellular organelle targeting or deliver genes to the peri-nucleus but randomly distributes them in the cell (Figure 2B, Figure 7A).

Figure 7.

Figure 7

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High levels of Lipo-Par transfection are independent of mitosis. (A) Schematic of the distribution of the microtubules and the ER during mitosis showing that Lipo-Pars are moving along the microtubules quickly to deliver genes to the nucleus. (B,C) pEGFP transfection when mitosis was inhibited by PTX (1 μg/mL). The scale bar represents 500 μm. The experiment was done three times. (D) Colocalization of the Lipo-Pars and the ER when mitosis was inhibited by PTX (1 μg/mL). The scale bar represents 50 μm. (E,F) Results from the experiments conducted to determine whether the Lipo-Pars or Lipo-Nons disrupted the cell cycle. Double-stranded DNA was stained with propidium iodide (PI), and flow cytometry was performed according to the product specification (cell cycle and apoptosis analysis kit, Beyotime). The percentage of cells in G2 is shown in (F). The experiment was done once. (G) Colocalization of the Lipo-Pars to the ER when cells were in mitosis. The merged parts are yellow. The scale bar represents 50 μm. (H) When the cells are in mitosis, the DNA carried by the Lipo-Pars is more likely to enter the nucleus. In telophase, the blue stain of the nucleus appeared more intense, and more of the foreign FITC-DNA (red) had entered the nucleus due to the incompleteness of the nuclear membrane. The images were captured after the cells were treated with Lipo-Par/DNA complexes for 10 or 60 min. The scale bar represents 50 μm. (I) Results from the high-resolution analysis of the distribution of the FITC-DNA carried by the Lipo-Pars when cells were in telophase. The plasmid coding mCherry-(calreticulin and KDEL) was transfected using Lipofectamine 2000. DNA is green, and the ER is red. Representative parts of the cells are enlarged. The scale bar represents 20 μm.

The more liposome/DNA complexes were internalized, the longer they stayed in the cell and the greater the likelihood that the DNA entered the nucleus. In the efflux experiment, we found that when the concentration and incubation time were controlled under the same conditions, the amount of intracellular DNA delivered by the Lipo-Pars was significantly greater, 7.0-fold, than that of the Lipo-Nons after the liposome/pEGFP complexes were internalized for 8 h. The DNA amount in the Lipo-Par group decreased to 57.8% and 40.4% of the original level (8 h) at 24 and 48 h, respectively, but this amount was still higher than the amount in the Lipo-Non group (which dropped to 20.8% and 15.2% of the original (8 h), respectively, at 24 and 48 h) (Figure 2D,E). These results demonstrate that the modification of pardaxin not only enhanced cationic liposome delivery of DNA but also prolonged the retention time of the internalized DNA.

Nonlysosomal caveolin-mediated endocytosis and increased DNA retention time in cells may be two critical reasons that the gene vector Lipo-Par can protect genes from damage and transfect them efficiently.

Lipo-Pars Are Actively Targeted to the ER

To investigate the intracellular transport pathway taken by the Lipo-Pars, we examined the release of the DNA from the liposome/DNA complexes over time, the colocalization of the liposomes with lysosomes, and the colocalization of liposomes and DNA with ER. Compared to the Lipo-Nons, the DNA combined with the Lipo-Pars (modified with pardaxin) was released at a rapid rate from the complexes, and the DNA began to enter the nucleus 2 h post-transfection (Figure 3A, right). The DNA delivered by the Lipo-Nons, in contrast, was still trapped in the complexes 4 h after transfection (Figure 3A, left). We hypothesize that the inefficient gene release of the DNA from the Lipo-Nons leads to DNA aimlessly circulating in the cytoplasm, which makes it easily destroyed by the acidic environment of the endosomes/lysosomes or through direct efflux. We used membrane-permeable fluorescent chemical probes to label the lysosomes and the ER separately. In the colocalization experiment of the liposomes and lysosomes, we found that the Lipo-Nons and the lysosomes were colocalization at a high rate at 2 h; in the case of the Lipo-Pars, although they also partially colocalized with the lysosomes, the colocalization signal was significantly weaker than that of the Lipo-Nons. These results suggested that, due to the diversity of the Lipo-Par internalization pathways, the Lipo/DNA complexes internalized via clathrin-mediated endocytosis were subsequently introduced to the lysosomes and that via caveolin-mediated endocytosis were able to successfully avoid the lysosomal pathway (Figure 3B). Since the cationic polypeptide pardaxin targets the ER, we hypothesized that the Lipo-Pars, which were covalently bound to pardaxin, may also have the ability to target the ER. We found that the Lipo-Pars and the ER showed significant colocalization at 8 h, while the colocalization of the Lipo-Nons and the ER was not obvious (Figure 3C). Additionally, a large amount of DNA carried by the Lipo-Pars had entered the nucleus at 8 h, while the remaining DNA was colocalized in or near the ER, a finding that was consistent with that shown in Figure 3A. This result indicated that the Lipo-Pars can deliver DNA to the ER and rapidly release the DNA from the complexes within the ER. The isolated DNA may enter the nucleus later by taking advantage of the intimate relationship between the ER membrane and the nuclear envelope.

Figure 3.

Figure 3

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Lipo-Pars are actively targeted to the ER and release DNA. (A) DNA release was observed at 1, 2, 4, and 6 h. DiD-labeled liposomes are green, FITC-labeled DNA is red, and the nucleus is dyed by Hoechst 33342. Representative single cells are enlarged. Compared to that released by the Lipo-Nons, DNA was released from the Lipo-Par faster and tended to merge with the nucleus within 6 h, according to the images. The scale bar represents 100 μm. (B) Colocalization of the liposomes and the DNA with lysosomes. Cells were incubated with Lipo-Pars (green) or Lipo-Nons (green) for 1.5 h at 37 °C, and the live cells were incubated with LysoTracker (red) (Beyotime Biotechnology) for an additional 20 min at 37 °C. The maximum excitation wavelength of the LysoTracker was 577 nm, and the maximum emission wavelength was 590 nm. Live-cell imaging was performed using an inverted fluorescence microscope (AIR, Nikon, Japan). The scale bar represents 50 μm. (C) Colocalization of the Lipo-Pars and Lipo-Nons with the ER. Cells were incubated with Lipo-Pars or Lipo-Nons for 7.5 h at 37 °C, and then, the fixed cells were incubated with the ER-tracker (Beyotime Biotechnology) for an additional 20 min at 37 °C. The maximum excitation wavelength of the LysoTracker is 587 nm, and the maximum emission wavelength is 615 nm. Live-cell imaging was performed using inverted fluorescence microscope (AIR, Nikon, Japan). The scale bar is represents 100 μm. (D) Results from the high-resolution colocalization analysis of the liposomes with the ER. Plasmid coding mCherry-(calreticulin and KDEL) was transfected using Lipofectamine 2000. Liposomes (10 μg/mL) were allowed to internalize for 2 h at 37 °C. Cells were fixed with 4% paraformaldehyde and observed using high-resolution laser confocal microscopy (LSM 880 with Airyscan, Carl Zeiss Jena, Germany). Representative parts of the cells are enlarged. Multichannel photos were captured on the same focal plane. (E) Results from the high-resolution colocalization analysis of the DNA-transfected Lipo-Pars and Lipo-Nons with the ER. The ER (red) was marked using the same method as (D). Nonfluorescent liposomes and FITC-DNA (green) complexes were generated and transfected for 2 h. Lipo-Par or Lipo-Non to DNA = 6:1 (mass ratio). Representative parts of the cells are enlarged. Multichannel photos were captured on the same focal plane. (F) Results from the 3D high-resolution colocalization analysis of Lipo-Non/Lipo-Par (green) with the ER (red). The merged parts are yellow. The scale bar represents 20 μm.

To observe the relative positions of the ER, liposome, and DNA more clearly, we used high-resolution laser confocal microscopy imaging to observe treated cells. We labeled the ER with a plasmid encoding mCherry-tagged calreticulin in live cells, and the labeled cells were incubated with DiD-labeled Lipo-Pars or Lipo-Nons for 2 h. It was found that the Lipo-Pars were evenly distributed in the ER, while the Lipo-Nons were distributed in the periphery of the ER. It was more interesting that some of the Lipo-Pars were located at the junction of the ER and the nucleus (Figure 3D), which was further confirmed by three-dimensional cell imaging (the yellow fluorescence signal represents colocalization of the ER with the Lipo-Pars) (Figure 3F). As a result, the internalized DNA carried by the Lipo-Pars has a greater tendency to localize in the peri-nuclear ER, while the Lipo-Nons simply delivered the DNA to the periphery of the ER, which was a distance from the nucleus (Figure 3E). This finding indicates that, through the unique function of pardaxin, the DNA can follow the Lipo-Pars to the ER, where the DNA is separated from the Lipo-Pars and enters into the nucleus possibly via the contiguous ER–nucleus membrane.

Our study clearly demonstrates that, compared to Lipo-Non/DNA complexes, Lipo-Par/DNA complexes released DNA more quickly after internalization, possibly due to the ER-targeting ability of the Lipo-Pars.

To further investigate how the Lipo-Pars targeted the ER, we used brefeldin A (BFA) (which inhibits vesicle transport from the ER to the Golgi apparatus)30−33 and nocodazole (which inhibits the transport of substances from the Golgi to the ER) to pretreat MCF-7 cells for 30 min, and then, we examined the ER targeting capability of the Lipo-Pars, using untreated cells as controls. As a result, the DiD-labeled Lipo-Pars were more concentrated in the BFA-treated cells compared with the control group, and in cells treated with nocodazole and BFA or nocodazole alone, the Lipo-Pars were distributed more randomly in the cytoplasm (Figure S4A,B). No significant differences were found between the Lipo-Non groups treated with BFA, nocodazole and BFA, or nocodazole and the control group. These results indicated that most of the Lipo-Pars were directly targeted to the ER after entering the cell, while some Lipo-Pars reached the ER through the Golgi-ER transport pathway. In addition, the Lipo-Pars already present in the ER can also be expelled via the ER-Golgi pathway.

Effect of ER Stress on Gene Transfection

ER stress (ERS) refers to the response to external stimulation that induces various downstream signaling cascades, many of which are closely related to cell survival and apoptosis.34,35 The ER-targeted distribution of the Lipo-Pars after internalization will inevitably disturb ER homeostasis and induce an ERS response. The transcription factor C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducing gene 153 (GADD153), is an ER stress marker protein.36,37 It has been reported that pardaxin, as a cationic peptide that targets the ER, caused ERS.12,38 We found that the pardaxin-labeled liposome, Lipo-Par, also caused an increase in the CHOP protein (Figure 4G). To explore the effects of ERS level on Lipo-Par-mediated gene transfection, we stimulated cell ERS with BAPTA (a calcium ion chelating agent), brefeldin A (ERS inducer), or KIRA6 (an ERS inhibitor, which inhibit IRE1α RNase kinase), and then incubated the cells with the liposome/pEGFP complexes.

Figure 4.

Figure 4

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Effect of ER stress on gene transfection. (A–C) Results from the test used to determine whether intracellular calcium level changes affected gene transfection. In (A), the cells were pretreated with 10 μM BAPTA-AM, a cell permeable calcium chelating agent, for 30 min, followed by incubation with liposome/DNA complexes for 2 h at 37 °C. In (B), the cells were pretreated with 5 μM or 10 μM BAPTA-AM for 30 min and then transfected with pEGFP. After transfection, the cells were incubated with 5 μM or 10 μM BAPTA-AM for an additional 22 h. In (C), the results from the quantitative analysis of (B) using ImageJ are shown. The scale bar of (A) represents 200 nm, and the scale bar of (B) represents 500 μm. The experiment was done three times. (D) Uptake of the liposomes when the intracellular calcium level was disrupted. Cells were pretreated with 10 μM BAPTA-AM for 30 min, followed by incubation with Lipo-Pars or Lipo-Nons (green) for 2 h at 37 °C. After liposome uptake, the cells were incubated with BAPTA-AM for an additional 1, 4, 10, and 22 h. The scale bar represents 100 μm. (E) Results from experiments used to determine whether ER stress affected gene transfection. Cells were pretreated with BFA (2 μg/mL) and transfected for 2 h. The scale bar represents 500 μm. The experiment was done three times. (F) Results from experiments used to determine how weakened ER stress affected gene transfection. Cells were pretreated with KIRA6 (5 μM), an inhibitor of IRE1α RNase kinase, and transfected for 2 h. The experiment was done three times. (G) Western blot was used to quantify C/EBP-homologous protein (chop/GADD153). Cells were incubated with Lipo-Nons or Lipo-Pars for 2 h. The experiment was done once.

It was found that in the cells pretreated with BAPTA-AM (5 μM), the GFP expression mediated by the Lipo-Pars and the Lipo-Nons was enhanced significantly compared to the GFP expression in the untreated cells (Figure 4B,C). Increasing the BAPTA-AM concentration further, from 5 μM to 10 μM, did not significantly increase the GFP expression mediated by the Lipo-Pars, indicating that 5 μM BAPTA-AM pretreatment has already contributed to the saturation of the transfected genes. To exclude the effects of cell uptake, we investigated whether BAPTA-AM affected the internalization of the liposomes, and we found that BAPTA-AM pretreatment had no significant influence on the uptake or intracellular localization of either the Lipo-Pars or the Lipo-Nons (Figure 4D), which means that the higher efficiency was not due to enhanced liposome uptake. However, cells treated with BAPTA-AM did not have a significantly different level of ssDNA internalization and/or ssDNA release (Figure 4A). In contrast, CaCl2 treatment to increase the number of cytoplasmic calcium ions, or heparin treatment to block IP3R39,40 suppressed GFP expression (Figure S5A,B). It was reported that the ERS response can induce ER membrane expansion,41 which most likely contributes to ameliorating the ERS. Therefore, we assume that membrane expansion induced by ERS also increased the intimate contact area of the ER membrane with the nuclear envelope, which may have provided more opportunities for the ER-localized DNA carried by the Lipo-Pars to enter the nucleus easily. BFA is usually used to stimulate ERS, and we found that the gene transfection mediated by the Lipo-Pars was also enhanced in the BFA-pretreated cells (Figure 4E). Conversely, inhibiting IRE1α-XBP1 signaling pathway (one of ERS pathways) with KIRA6 could reduce GFP fluorescence intensity significantly (Figure 4F). In addition, the GFP expression mediated by the Lipo-Nons in the BFA-pretreated group was enhanced, which surprised us, leading us to speculate that the deliberate induction of ER stress might have had a positive effect on the gene transfection (Figure 5).

Figure 5.

Figure 5

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ERS enhances pGFP transfection. When cells were stimulated with ERS-related reagents (e.g., BAPTA-AM or BFA), the non-ER-targeting liposomes, Lipo-Nons, achieved higher transfection efficiency.

Intracellular Transport of the Lipo-Pars

Colchicine can inhibit the polymerization of microtubules (MT). In the internalization experiments, MCF-7 cells were incubated with DiD-labeled Lipo-Pars after pretreatment with 25 μM colchicine. The results showed that the colchicine-induced microtubule depolymerization did not affect the internalization of the Lipo-Pars, but the Lipo-Pars tended to distribute randomly in the cytoplasm instead of distributing centrally in the peri-nucleus ER (Figure 6A). Interestingly, the colchicine-induced microtubule depolymerization resulted in a significant decrease in Lipo-Par-mediated gene transfection efficiency (from 45.5% to 6.3%), whereas for the Lipo-Nons, the efficiency decrease was more modest (from 1.1% to 0.6%) (Figure 6B,C,D). Three-dimensional imaging showed that the Lipo-Pars (green in Figure 6E; pink in Figure 6F) and microtubules (red in Figure 6E,; green in Figure 6F) were obviously colocalized, as shown by the bright yellow (Figure 6E) or white (Figure 6F) fluorescing parts. These results demonstrate that microtubules played an important role in the gene delivery mediated by the Lipo-Pars. It is quite possible that the Lipo-Pars carry DNA along the microtubules to the ER, thereby enabling DNA to entry the nucleus. Once the microtubules are depolymerized, it is difficult for the Lipo-Pars to accumulate in the ER, but instead diffusing throughout the cytoplasm, which resulted in an apparent decrease in the transfection efficiency. Further, we found that some Lipo-Pars also colocalized with microfilaments (Figure 6G), suggesting that the Lipo-Pars also were intracellularly transported along microfilaments.

Figure 6.

Figure 6

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Microtubules play a critical role in the efficient internalization of the Lipo-Pars. (A) Distribution of the liposomes when microtubules were depolymerized by 25 μM colchicine. Liposomes (green) are distributed randomly throughout the cells. The scale bar is 20 μm. (B–D) Results from the gene transfection comparison when the microtubules were depolymerized. Cells were pretreated with 25 μM colchicine, followed by pEGFP transfection for 2 h at 37 °C. After transfection, the cells were cultured for an additional 22 h. Flow cytometry was used to analyze transfection efficiency quantitatively, and the results are shown in (C) and (D). The scale bar of (B) is 500 μm. The experiment was done once. (E,F) 3D high-resolution analysis of the colocalization of the Lipo-Pars with the microtubules (MT). Plasmid coding mEmerald-tubulin was transfected using Lipofectamine 2000 and the Lipo-Pars (10 μg/mL) were allowed to internalize for 1 h (E) or 2 h (F) at 37 °C. Cells were fixed with 4% paraformaldehyde and observed using high-resolution laser confocal microscopy (LSM 880 with Airyscan, Carl Zeiss Jena, Germany). Representative parts of the cells are enlarged. Multichannel photos were taken on the same focal plane. The scale bar is 50 μm. (G) High-resolution colocalization analysis of the Lipo-Pars and microfilaments. Polymeric F-actin (red) was dyed using phalloidin–tetramethylrhodamine B isothiocyanate (Sigma-Aldrich) following the protocol. Multichannel photos were taken on the same focal plane. The scale bar represents 50 μm.

The cell cytoskeleton, including microtubules and microfilaments, facilitates the high-speed transport of vesicles, during which kinesin (which is mainly responsible for plus-end transport) and dynein (which is mainly responsible for minus-end transport) matter greatly. Microtubules are responsible for the long-distance transport of vesicles between the minus ends at the cell center and the plus ends directed to the cell periphery, and the microfilaments are responsible for the short-distance transport of vesicles.42,43 As the figures above show, Lipo-Pars undergo self-determined localization by taking advantage of the exquisite cytoskeleton, and this method is very similar to that by which viruses are transported; therefore, we defined the Lipo-Par as a virus-like nonviral gene vector.

Cell Mitosis and Gene Transfection

Cationic liposomes are thought to make use of cell mitosis to achieve efficient gene transfection.44−46 However, similar to the Lipo-2000s and Lipo-Nons, the Lipo-Pars did not interrupt normal mitosis, a finding that was indicated by the flow cytometry results (Figure 7E,F), indicating that the Lipo-Pars do not achieve high efficiency by promoting cell mitosis. To investigate the effect of impaired mitosis on Lipo-Par-mediated gene transfection, cells were pretreated with paclitaxel (PTX) and then incubated with the liposome/pEGFP complexes, and the GFP fluorescence was detected. The results showed that the cells treated with PTX for 30 min were significantly retained at the G2 phase, indicating that mitosis was inhibited (Figure 7E,F), and the cell viability was 72.45% when the cells were pretreated with 1 μg/mL PTX for 30 min (Figure S5C). In addition, it was found that the Lipo-2000- and Lipo-Par-mediated GFP transfection efficiency was reduced due to the inhibited mitosis (Figure 7B,C), suggesting that, although these nonviral vector genes did not achieve efficient transfection by promoting mitosis, impaired mitosis can significantly reduce their transfection efficiency. Since the Lipo-Pars are capable of delivering foreign genes to the peri-nucleus ER, these genes are highly accessible to the nucleus during mitosis. By high-resolution laser confocal microscopy, it was found that, when cells were in mitosis, the Lipo-Pars and the exogenous DNA carried by the Lipo-Pars tended to concentrate in the gap between the two daughter cells (Figure 7G,I), and the DNA carried by the Lipo-Pars entered the nucleus more quickly during mitosis (Figure 7H). Additionally, when the cells were pretreated with PTX, the gene transfection efficiency mediated by the Lipo-2000s declined more significantly than that mediated by the Lipo-Pars, which emphasizes that the Lipo-2000s depend on cell mitosis to a greater extent, compared to the Lipo-Pars, to achieve transfection (Figure 7B,C). This finding might be contributed to the unique intracellular transport method of the Lipo-Pars: they still deliver genes to ER along microtubules in the PTX-treated cells (Figure 7D).

ER-targeting liposomes have excellent position advantages. It is well-known that the nuclear envelope consists of inner and outer membranes, as well as nuclear pore complexes, and that the outer nuclear membrane is connected to the ER membrane. Furthermore, the nuclear envelope depolymerizes during prophase and reconstitutes with the ER membrane during anaphase, and there is close contact between the nuclear components and the microtubules and microfilaments.15,16,47 The lipo-Pars linked to the ER-targeting peptide pardaxin accumulated on the peri-nuclear ER membrane, which created more opportunities for the DNA they carry to enter the nucleus regardless of whether the cell was undergoing mitosis or not. This characteristic of the Lipo-Par ER-targeting may be one of the critical reasons that the Lipo-Pars achieve efficient gene transfection (Figure 7A).

Discussion

Although questioned over its safety, gene therapy still has good application prospects in the treatment of diseases such as tumors and cardiovascular diseases because of its relatively high efficiency and specificity. As of 2017, a total of 1688 cancer patients were treated clinically by gene therapy, accounting for 65% of the indications addressed by gene therapy clinical trials.1 Since the biosafety of liposomes is superior to that of viral vectors, it has had good momentum in clinical applications. However, with the low transfection efficiency still being the bottleneck in the laboratory and the clinic, its development for application remains greatly limited.

Referring to the intracellular transport mechanism of highly efficient viral infections, we synthesized cationic peptide pardaxin, which was used to obtain DSPE-PEG2000-Par, through a condensation reaction with DSPE-PEG2000-NH2, and we additionally synthesized the cationic liposome Lipo-Par.

Compared to non-ER-targeting vectors, Lipo-Par is a nonviral vector that mimics the viral infection mechanism to transfect exogenous genes more efficiently into cancer cells of multiple lineages, even surpassing the efficiency of Lipofectamine 2000 (Figure 1D,E). The mechanism of the Lipo-Par-mediated efficient transfection may be manifested by the following findings: (1) after internalization by caveolin-mediated endocytosis, the Lipo-Pars are transport along microtubules and microfilaments, preventing the genes from encountering the acidic degradation of the lysosomes; (2) the Lipo-Pars deliver gene to the peri-nuclear ER rapidly, giving genes closer access to nucleus; (3) ER stress has a positive effect on gene transfection; and (4) the ER-nucleus pathway, based on the intimate relationship of the ER membrane and nuclear envelope, promotes gene entry into the nucleus for successful transfection.

Since the ER stress response can be stimulated when Lipo-Pars are enriched in the ER, we speculate that the ER membrane expanses due to the ER stress, which increases the contact area with the nucleus, creating more opportunities for exogenous genes to enter the nucleus. Although mitosis increases gene delivery efficiency, as verified in many previous studies, our study suggests that the gene transfection mediated by the Lipo-Pars is not completely dependent on mitosis. During interphase, Lipo-Pars showed excellent transfection efficiency in several types of human malignant cells without interfering with normal mitosis.

Although the virus-like nonviral gene vector Lipo-Par features innovations in terms of transfection efficiency and delivery mechanism, the molecular mechanism of its intracellular transport process remains to be investigated; for example, how do they transfer to the microtubules after endocytosis? How do they bind to the motor protein? What happens in the ER in addition to the mild increase in ER stress? Do locally high concentrations of calcium ions in the ER promote rapid release of genes from the vector rapidly? Do exogenous genes enter the nucleus through the nuclear pore complexes in a manner similar to that of viruses, or by membrane exchange or membrane flow? Although there are still many unknown mechanisms, our research may provide another useful reference for the development of efficient nonviral gene vectors.

Conclusions

Lipo-Par, an SV40 virus-like nonvirus cationic liposome modified with the cationic peptide pardaxin, was found to possess a novel mechanism that efficiently transfects exogenous genes. This unique mechanism can result in the prevention of lysosomal capture, active targeting to the ER along the cell cytoskeleton, stimulated mild ER stress, and leveraged use of the intimate relationship between the ER membrane and the nuclear envelope (see abstract graphic), actions that are similar to those of the virus infection mechanism. Therefore, we speculate that the ER may be a new and effective targeting point for nonvectors to achieve the desired transfection efficiency.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81973246 and 81573365) and Basic Public Welfare Research Project of Zhejiang Province, China (LGF18H300004).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.9b01052.

  • Synthesis and characterization; Lipo-Par internalization mechanism; ER-to-Golgi and the Golgi-to-ER transport of the liposomes; effects of calcium ions on gene transfection (PDF)

The authors declare no competing financial interest.

Supplementary Material

oc9b01052_si_001.pdf (1.5MB, pdf)

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