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The gene transfection and endocytic uptake pathways mediated by PEGylated PEI-entrapped gold nanoparticles
⁎Corresponding authors at: Key Laboratory of Science & Technology of Eco-Textile, Ministry of Education, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China (X. Shi and X. Cao). xshi@dhu.edu.cn (Xiangyang Shi), caoxy_116@dhu.edu.cn (Xueyan Cao)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
The development of gene vectors is the vital step in gene therapy. The cationic polymer polyethylenimine (PEI) is widely applied as an efficient and low cost nonviral gene vehicle. However, its further practical applications in gene therapy are limited due to its high cytotoxicity. To obtain safe and efficient gene vectors, hydrophilic polyethylene glycol (PEG) has been conjugated onto its surface due to its good biocompatibility, and the PEGylated PEI was used as a template to entrap gold nanoparticles (Au NPs) with different Au atom/PEI molar ratios (25:1, 50:1, 100:1, and 200:1, respectively). The formed PEGylated PEI-entrapped Au NPs (PEG-Au PENPs) and their cytotoxic effects as well as ability to transfect plasmid DNA (pDNAs) to HeLa cells were analyzed using Cell Counting Kit-8 (CCK-8) assay, flow cytometry and confocal microscopic imaging. To further understand cell internalization pathway of PEG-Au PENPs, several pharmacologic inhibitions of endocytic pathways were conducted. Our results revealed that the PEG-Au PENPs were not only able to transfect pDNAs into cells with decreased cytotoxicity, but also showed high transfection efficiency compared with PEI alone. The cellular uptake data indicated that the clathrin-mediated endocytosis is the main pathway in the internalization of the formed polyplexes. These findings suggested that the developed PEG-Au PENPs may serve as a safe gene carrier with non-compromised DNA transfection efficiency and promote the further development of efficient and safe gene delivery strategies based on nanoparticles.
Keywords
PEGylated polyethylenimine
Gold nanoparticles
Transfection efficiency
Cyctotoxicity
Endocytic pathways
1 Introduction
There has been an upward trend associated with the incidence of various gene-related diseases, so it is urgent to find a low cost and effective therapeutic method to treat these diseases. Gene therapy brings about a new prospect to these diseases, and has been known to be one of the main therapies of the acquired diseases (Qasim et al., 2009). Gene therapy is a technology transferring exogenous gene such as plasmid DNA (pDNA) or small interfering RNA (siRNA) into the target cells to treat gene-mediated diseases (Verma and Weitzman, 2005). Finding an excellent vector which can transfer therapeutic genes into cells safely and efficiently remains a challenge (Fant et al., 2010). Although viral vectors usually have a strong transfection ability, they display high cytotoxicity, carcinogenic and immunogenicity (Kim et al., 2004; Navarro and Tros de ILarduya, 2009). Nonviral, especially polycationic nuclear acids delivery systems have attracted much attention due to the characteristics of easy preparation, simple modification, relative safety and high genetic loading capacity (Cao et al., 2017; Kim et al., 2009).
Recently, a lot of nanomaterials have been used in the diagnostics, for example, Tang et al. reported several nanomaterials-based photoelectrochemical biosensing platform for cancer detection (Qiu et al., 2018; Shu et al., 2018; Shu and Tang, 2017; Zhang et al., 2018; Zhou et al., 2018). Many cationic polymeric carriers have also been used in gene delivery, such as polyethylenimine (PEI) (Godbey et al., 1999; Kunath et al., 2003), lipids (Lv et al., 2006; Pedroso De Lima et al., 2001), polylysine (Cai et al., 2015; Zhu et al., 2014), poly (amidoamine) (PAMAM) dendrimers (Shakhbazau et al., 2010; Xiao et al., 2013), and chitosan (Chan et al., 2007; Köping–Höggård et al., 2004). In addition, the nano-vesicle-encapsulated plasmonic Au NPs were functionalized with a double-stranded DNA which was used in wild-type p53(WTp53) recognizes (Qian et al., 2016); a two-stage dissociation nanoparticle system based on multi-functionalized polydopamine-coated gold nanoparticles was used to investigate the complex interaction between various cancer biomarkers and to describe the cancer biomarker-synergic networks in single cells (Qian et al., 2017). PEI with abundant surface amine groups can compact DNA efficiently and has shown immense efficacy, being the golden standard of the gene carriers (Boussif et al., 1995; Lungwitz et al., 2005). However, the existence of PEI amine-induced cytotoxicity leads to limitations in its further clinical applications.
In order to reduce the cytotoxicity, properly modifying the PEI architecture and surface functionalities has been attempted (Kircheis et al., 2001; Ogris et al., 1999). For example, Wang et al. used PEI-modified Pluronic copolymers (PCMs) as a gene delivery carrier with low cytotoxicity (Wang et al., 2012). Kichler et al. showed that the polyethylene glycol (PEG)-grafted PEI had an 8% increase in the cell viability when compared with PEI alone (Kichler et al., 2002). Besides, our previous study reported that PAMAM dendrimer-entrapped Au NPs (Au DENPs) showed a higher gene delivery efficacy and lower cytotoxicity than PAMAM dendrimers alone, which could be attributed to the fact that the presence of Au NPs can render the dendrimers with three-dimensional (3D) shape (Shan et al., 2012). Due to the fact that the molecular structure of PEI is similar to PAMAM dendrimers, we logically speculate that PEI can also be analogously modified for improved gene therapy.
Endocytic uptake pathways are considered as an essential process for further understanding the mechanism of gene delivery (Alex and Sharma, 2014; Doherty and McMahon, 2009). In general, endocytic pathways responsible for the uptake of nanomaterials fall into three major categories: the receptor-mediated endocytosis, the receptor-independent pathway and macropinocytosis (Pereira et al., 2015; Sevimli et al., 2015; Zhang et al., 2011). The receptor-mediated endocytosis is also known as clathrin-mediated endocytosis. The receptor-independent pathway can be further subdivided into caveolin-mediated endocytosis, clathrin-independent endocytosis, clathrin- and caveolin-independent endocytosis. It is important to know the endocytosis pathway during the gene delivery process.
In the present study, we prepared the PEGylated PEI-entrapped Au NPs (PEG-Au PENPs) as vectors for gene transfection and endocytosis. Aminated branched PEI is used as a template to synthesize PEG-Au PENPs with various Au atom/PEI molar ratios. Cell Counting Kit-8 (CCK-8) was used to test the cytotoxicity of PEG-Au PENPs. The ability of the PEG-Au PENPs to compact pDNA was tested by gel retardation assay. Then, the gene transfection efficiency was tested by using PEG-Au PENPs as a gene delivery vector to transfect both luciferase (Luc) reporter gene and enhanced green fluorescent protein (EGFP) gene to HeLa cells. The intracellular uptake capacity and intracellular trafficking of the carrier/pDNA polyplexes were studied through flow cytometry and confocal microscopic imaging. In addition, the endocytic uptake pathways of the PEG-Au PENPs in HeLa cells were also characterized using dynasore, nystatin and chlorpromazine through flow cytometry. Since macropinocytosis is a macrophage-centered endocytosis pathway (Kerr and Teasdale, 2009; Lim and Gleeson, 2011; Swanson and Watts, 1995), the current study is principally focused on both the receptor-mediated and receptor-independent pathways.
2 Experimental details
2.1 Materials
Aminated branched PEI (molecular weight = 25,000) and ethidium bromide (95%) were purchased from Aldrich (St. Louis, MO). Agarose was from Gene Tech (Shanghai, China). CCK-8 was from 7sea Shanghai Futai Biotech Co., ltd. (Shanghai, China). Luc assay kit was purchased from Promega Corporation (Madison, WI). Bicinchoninic acid (BCA) protein quantitation kit was purchased from Applygen technologies Inc. (Beijing, China). Dynasore, nystatin and chlorpromazine were from MedChem Express (Monmouth Junction, NJ). All of the cell culture flasks and plates were from NEST Biotechnology (Shanghai, China).
2.2 Preparation and characterization of PEG-Au PENPs
PEG-Au PENPs were synthesized according to our previous work (Li et al., 2016; Zhou et al., 2014). The PEG-Au PENPs were prepared by Sodium borohydride (NaBH4) reduction chemistry using PEI as a template with gold salt (HAuCl4)/branched PEI molar ratio at 25:1, 50:1, 100:1, and 200:1, respectively. First, the formed PEI·NH2-mPEG conjugate was characterized by 1H NMR (Fig. S1, Supporting Information) and MALDITOF mass spectral analysis (Fig. S2, Supporting Information). The formed PEG-Au PENPs were characterized by UV–vis spectroscopy (Fig. S3, Supporting Information) using a Lambda 25 UV–vis spectrophotometer (Perkin Elmer, Boston, MA). A JEOL 2010F analytical electron microscope (JEOL, Tokyo, Japan) was used to characterize the size of the PEG-Au PENPs at 200 kV via transmission electron microscopy (TEM) imaging (Fig. S4, Supporting Information). The crystalline structure of Au core NPs was further validated by selected area electron diffraction (SAED) (Fig. S5a, Supporting Information). Energy dispersive spectroscopy (EDS) analysis of the PEG-Au PENPs confirmed the presence of gold element (Fig. S5b, Supporting Information). The synthesized PEG-Au PENPs are quite stable in water, PBS, and cell culture medium and don’t precipitate for at least 3 months. For simplicity, the PEG-Au PENPs with the Au atom/PEI molar ratio at 25:1, 50:1, 100:1, and 200:1 were represented as L25, L50, L100, and L200, respectively in our naming system.
Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.arabjc.2018.06.009.
PEG-Au PENPs were synthesized according to our previous work (Li et al., 2016; Zhou et al., 2014). The PEG-Au PENPs were prepared by Sodium borohydride (NaBH4) reduction chemistry using PEI as a template with gold salt (HAuCl4)/branched PEI molar ratio at 25:1, 50:1, 100:1, and 200:1, respectively. First, the formed PEI·NH2-mPEG conjugate was characterized by 1H NMR (Fig. S1, Supporting Information) and MALDITOF mass spectral analysis (Fig. S2, Supporting Information). The formed PEG-Au PENPs were characterized by UV–vis spectroscopy (Fig. S3, Supporting Information) using a Lambda 25 UV–vis spectrophotometer (Perkin Elmer, Boston, MA). A JEOL 2010F analytical electron microscope (JEOL, Tokyo, Japan) was used to characterize the size of the PEG-Au PENPs at 200 kV via transmission electron microscopy (TEM) imaging (Fig. S4, Supporting Information). The crystalline structure of Au core NPs was further validated by selected area electron diffraction (SAED) (Fig. S5a, Supporting Information). Energy dispersive spectroscopy (EDS) analysis of the PEG-Au PENPs confirmed the presence of gold element (Fig. S5b, Supporting Information). The synthesized PEG-Au PENPs are quite stable in water, PBS, and cell culture medium and don’t precipitate for at least 3 months. For simplicity, the PEG-Au PENPs with the Au atom/PEI molar ratio at 25:1, 50:1, 100:1, and 200:1 were represented as L25, L50, L100, and L200, respectively in our naming system.
Supporting information
Supporting information EXPERIMENTAL DETAILS (Agarose gel retardation assay; Dynamic light scattering (DLS) and zeta potential measurements; Statistical analysis), 1H NMR; MALDITOF mass spectral; UV−vis spectra; TEM images and size distribution histograms; SAED and EDS of the PEG-Au PENPs as Supporting Information.2.3 Cytotoxicity assay
The cytotoxicity of various carriers was measured by CCK-8 assay of HeLa cells upon treatment with the tested carriers at various concentrations. HeLa cell suspension (8000 cells/well) was seeded in a 96-well plate and cultivated in 100 µL Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin for each well under 37 °C and 5% CO2 for 24 h. Subsequently, the medium was exchanged with serum-free DMEM containing vectors with a final concentration ranging from 50 to 3000 nM. The cells were further incubated for 24 h under the same condition. Thereafter, 10 μL of CCK-8 solution was added into each well and the cells were incubated for 4 h. Finally, each sample with five replicates (n = 5) was measured using a microplate reader (BioTek, ELX800, USA) to record the absorbance at 450 nm. Phosphate buffered saline (PBS) was used as control.
2.4 Polyplexes preparation
PEG-Au PENPs/pDNA polyplexes were produced with several different mass ratios (the mass ratio of the PEI to the pDNA). Both carriers and pDNA were prepared with PBS buffer (pH 7.4) and then mixed together according to the varied mass ratios. The vector and pDNA solutions were vortexed gently for seconds and incubated for 25 min at room temperature to obtain the vector/pDNA polyplexes.
2.5 Gene transfection
HeLa cell suspension (4 × 104 cell/well) was dispensed in a 24-well plate and cultivated in an incubator under 37 °C and 5% CO2 overnight. Prior to transfection, medium in each well was replaced with 400 μL of serum-free DMEM when the cells reached 60–70% confluence. Then, a 100 μL of polyplex solution was added to each well of the plate and the cells were transfected for 4 h. Thereafter, the culture medium was exchanged with fresh medium with serum and incubated for another 24 h. The cells were harvested and Luc activities were tested according to the protocol of Promega’s Luc assay. The transfection efficiency (n = 3) of each sample was measured by relative light unit per milligram of total protein (RLU/mg). Non-transfected cells and cells transfected with naked pDNA were treated as negative controls. PEI was employed as a positive control. Polyplex solutions were prepared by mixing various vectors with Luc pDNA (1.0 μg/well) under mass ratios of 2:1, 4:1, and 6:1, respectively.
EGFP expression studies were also carried out to prove the gene transfection efficacy of PEG-Au PENPs. Similarly, HeLa cells were transfected with the polyplexes containing EGFP pDNA (1.0 μg/well) under mass ratios of 2:1, 4:1, and 6:1, respectively. After 24 h transfection, cells were observed using fluorescence microscope (Carl Zeiss Axio Vert.A1, Jena, Germany).
2.6 Cellular uptake and intracellular trafficking of PEG-Au PENPs/pDNA polyplexes
To study the intracellular uptake capacity of the various vector-DNA polyplexes, Cy3-labeled pEGFP DNA (1.0 μg/well) was mixed with different carriers, respectively. The intracellular uptake capacity was tested at the mass ratio of 4:1 by flow cytometry (BD FACS Calibur, USA). HeLa cell suspension (1 × 105 cell/well) was dispensed in a 6-well plate and cultivated in an incubator under 37 °C and 5% CO2 overnight. Prior to transfection, the medium was replaced with serum-free DMEM. Then the polyplex solutions were added to the plate and transfected for 4 h. Subsequently, the cells were harvested by trypsin and PBS. Finally the harvested cells were analyzed by flow cytometry.
Confocal laser scanning microscopy (CLSM) was employed to compare the dissimilarity between PEI and PEG-Au PENPs with regard to their applications as carriers in intracellular trafficking. HeLa cells were incubated with polyplexes (as described above) for 4 h. Then the cells were fixed with 5% glutaraldehyde for 15 min at 4 °C and DAPI was used to stain the cell nuclei for 7 min. Finally, cells were washed 3 times with PBS to eliminate the background signals and were observed by confocal microscopy (Carl Zeiss LSM700, Jena, Germany).
2.7 The endocytic profile of PEG-Au PENPs
In order to identify the portals of PEG-Au PENPs in HeLa cells, several chemical inhibitors were chosen to block specific endocytic pathways associated with the uptake of the tested nanoparticles (NPs). Briefly, 1 × 105 cells were seeded in a 24-well plate and cultivated overnight. Then the medium was replaced with the serum-free DMEM containing dynasore (60 μM) or nystatin (20 μg/mL), and the cells were incubated for 2 h at the same incubation condition. Later, the cells were reincubated with polyplexes (as described above) for 4 h in the presence of inhibitors. Regarding the chlorpromazine treatment, HeLa cells were treated with chlorpromazine (10 μM) for 30 min. Subsequently, polyplexes were added and the cells were reincubated for 4 h. The cells treated with the nanomaterials without inhibitors were used as a control. Thereafter, cells were analyzed using Flow Cytometry.
3 Results and discussion
3.1 Cytotoxicity of the vectors
According to our previous work, we successfully prepared the PEG-Au PENPs with different Au/PEI molar ratios (Zhou et al., 2014). To evaluate the cytotoxicity of the PEG-Au PENPs on HeLa cells, CCK-8 assay was performed before the gene transfection studies. As shown in Fig. 1. it is obvious that the cell viability displayed a concentration-dependent manner for PEG-Au PENPs and PEI. It means that with the increase of the materials concentration for all samples, the cell viability gradually reduces. It is noticeable that PEI alone is much more toxic than all the PEG-Au PENPs at higher concentrations. It may be due to the fact that the entrapment of Au NPs compensates the surface amine groups of PEI. After PEG modification, the number of the amine groups is further greatly decreased (Hou et al., 2015; Xiao et al., 2015). For tested NPs, it is interesting that the cytotoxicity of the carriers follows the order of PEI > L25 > L50 > L100 > L200 at a relatively higher concentration (above 1500 nM), since more PEI surface amines are needed to stabilize the entrapped Au NPs with the increment of the gold contents, leading to lower positive charge and lower cytotoxicity. Our results indicate that the PEG-Au PENPs are more likely to be safer gene transfection carriers than the PEI alone for gene delivery applications.
Cell viability assay of HeLa cells treated with different carriers under different concentrations (mean ± SD, n = 5).
3.2 Assessment of gene transfection ability
In view of the low toxicity of PEG-Au PENPs, we routinely assessed the gene transfection ability of PEG-Au PENPs. We used gel retardation assay to evaluate the genetic loading capacity of the PEG-Au PENPs before gene transfection. It is obvious that all PEG-Au PENPs were capable of compacting pDNA completely at a mass ratio of 2 or greater (Fig. 2). Thus, we chose the mass ratios of 2, 4 and 6 to perform the following studies. It is well known that the hydrodynamic sizes and zeta potential of polyplexes are also key parameters impacting the gene transfection. The results of hydrodynamic sizes demonstrated that most polyplexes formed have a size ranging from 150 to 300 nm at different mass ratios, which are regarded to be beneficial for gene transfection (Fig. 3a). (Conner and Schmid, 2003) It is worth noting that at a mass ratio of 4, the formed polyplexes with pDNA for all the carriers seem to be smallest in size (∼200 nm) in spite of the Au atom/PEI molar ratio. Because the polyplexes in this size range are regarded to be able to facilitate internalization by endocytosis, (Conner and Schmid, 2003) so at the mass ratio of 4, the polyplexes may perform well in gene delivery. For the PEG–Au PENPs carriers, the hydrodynamic diameter decreases with the increase of the Au atom/PEI molar ratio for the PEG–Au PENPs, however when the mass ratio is increased to 4:1 and 6:1, the diameter of the formed compounds remains almost the same. The results of zeta potential showed that the vector/pDNA polyplexes are all positively charged and reach about 25 mV, suggesting that polyplexes could have the good transfection capacity (Fig. 3b).
Electrophoretic mobility retardation assay of pDNA complexed with PEI (a), L25 (b), L50 (c), L100 (d), and L200 (e) under various mass ratios. Lane 1: DNA marker; lane 2: mass ratio = 0.5:1; lane 3: mass ratio = 1:1; lane 4: mass ratio = 2:1; lane 5: mass ratio = 4:1; lane 6: mass ratio = 6:1; lane 7: and mass ratio = 8:1.
Hydrodynamic particle size (a) and surface potential (b) of the polyplexes under three different mass ratios.
Gene transfection efficiency was evaluated through qualitative and quantitative assays. EGFP displays bright green fluorescence, which can be observed by fluorescence microscopy. Herein, PEG-Au PENPs prepared with different gold contents are used to transfect pDNA encoding EGFP protein to HeLa cells at the selected mass ratios for 24 h (Fig. 4). Fluorescence microscopic images revealed that cells transfected with L25/pDNA polyplexes presents more efficient EGFP expression than the other polyplexes at a mass ratio of 4:1. Comparing with the control groups without any treatment or incubation with pDNA alone, cells treated with polyplexes formed using all PEG-Au PENPs showed much higher EGFP expression. It is interesting to note that the EGFP gene expression using the PEG-Au PENPs as vectors is not enhanced with the gold loading in the PEGylated PEI, or with the size of the Au core NPs entrapped within the PEGylated PEI. For the PEG-Au PENPs prepared with the increased Au atom/PEI molar ratios, the formed Au NP cores have a larger size, which requires more PEI surface amines to stabilize the Au NPs, consequently resulting in a lower compaction ability to pDNA and gene delivery efficacy than those of L25 and PEI.
Fluorescence microscopic images (200×) of EGFP gene expression in HeLa cells using PEI (a), L25 (b), L50 (c), L100 (d) and L200 (e) vectors at the mass ratios of 2 : 1, 4 : 1, 6 : 1, respectively. PBS and pDNA are phase contrast images. Images were taken 24 h after transfection under similar instrumental conditions. Scale bar = 200 μm.
The gene delivery efficiency of the PEG-Au PENPs was further investigated by Luc gene expression. It is obvious that L25 enabled the highest protein production in cells among the PEG-Au PENPs and have a peak value at a mass ratio of 4:1 (Fig. 5). The Luc expression of L25 is very close to that of the PEI vector, at the proper mass ratio, which is in accordance with the EGFP activity assay result. These results strongly imply that PEG-Au PENPs with an appropriate composition have a low cytotoxicity but show a non-compromised efficiency of gene delivery. Taken together with the EGFP gene expression results, our findings indicated that PEG-Au PENPs with proper Au atom/PEI molar ratios may be used as a safe and efficient gene delivery carrier. In our recently published work (Xu et al., 2018), we reported that the PEGylated folate-conjugated Au DENPs with low immunogenicity for targeted gene delivery. In the present work, we used PEI as a template to synthesize PEG-Au PENPs for gene delivery. PEI can be easily developed using an AB-type monomer through a simple one-step reaction, which is strikingly different from the dendrimer synthesis through a time-consuming divergent or convergent technology (Zhou et al., 2014). Compared to dendrimers, PEI is cheap and has been considered the golden standard for the gene carriers (Boussif et al., 1995; Lungwitz et al., 2005).
Luciferase gene transfection efficiency of PEG-Au PENPs/DNA polyplexes in HeLa cells at mass ratios of 2:1, 4:1, and 6:1, respectively (mean ± SD, n = 3). Cells without treatment (None) and cells treated with vector-free pDNA were used as controls. Statistical differences between PEG-Au PENPs (L25, L50, L100, and L200, respectively) versus PEI at a mass ratio of 4:1 was compared and indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.
3.3 Cellular uptake and intracellular trafficking
In order to further address the underlying mechanism related to the PEG-Au PENPs for gene delivery, we attempted to study the difference of various vector/pDNA polyplexes with regard to their cellular uptake and colocalization process via flow cytometry and confocal microscopic imaging. Due to the fact that all tested carriers showed highest gene delivery efficacy at the mass ratio of 4:1 from gene transfection results, we decided to select the mass ratio of 4:1 as an optimal one for further study. Flow cytometry showed that around 96.44% and 91.80% of HeLa cells display Cy3-derived red fluorescence signal when PEI and L25 were as vectors, respectively (Fig. 6), while the proportions of the red fluorescent cells are 85.10%, 56.38% and 48.22% when L50, L100, and L200 were used as vectors, respectively. The cellular uptake of L25/pDNA polyplex is almost the same as that of PEI/pDNA polyplex, which is in accordance with the gene transfection results. Confocal microscopic images displayed the intracellular localization of each polyplex (Fig. 7). There was no red fluorescence signal observed in the cell cytoplasm for the group of naked DNA without vector, which suggested failure to be internalized by cells. In contrast, the vector/pDNA polyplexes, especially the PEI/DNA and L25/DNA polyplexes could be visualized inside the cells, showing red fluorescence signals. The red fluorescence intensity follows the order of PEI > L25 > L50 > L75 > L100. The result indicates that all the PEG-Au PENPs follows the same pathway as PEI in the intracellular trafficking. Overall, our studies confirmed the gene transfection results and suggested that L25 could be an efficient and safe gene vector.
Flow cytometry measurement of the intracellular uptake of carrier/Cy3-labeled pDNA polyplexes at the mass ratio of 4 in HeLa cells. (a) Control cells without treatment, (b) naked DNA without vector, (c) PEI/pDNA, (d) L25/pDNA, (e) L50/pDNA, (f) L100/pDNA, (g) L200/pDNA, and (h) the population distribution histograms of cells with Cy3 fluorescence within the selected gate. Transfection was performed at a dose of 1.0 μg/well of pDNA (mean ± SD, n = 3).
Confocal microscopy images of intracellular trafficking of the Cy3-labeled pDNA with carriers at the mass ratio of 4 in HeLa cells. (a) naked DNA without vector, (b) PEI/pDNA, (c) L25/pDNA, (d) L50/pDNA, (e) L100/pDNA, (f) L200/pDNA. Images were taken 4 h after transfection (red: Cy3-labeled pDNA; blue: DAPI stained cell nuclei). Scale bar = 10 μm.
3.4 Endocytic pathway study
It is very meaningful to further investigate the endocytic pathway of the vector for gene delivery (Hafez and Cullis, 2001; Xu and Szoka, 1996; Zabner et al., 1995; Zhou and Huang, 1994; Zuhorn et al., 2002). Zhang et al. reported that arginine-conjugated chitosan can be internalized by the caveolin-mediated endocytic pathways (Zhang et al., 2011). In another study, Au nanocage uptake in PC3 cells was proven to be mediated by clathrin-dependent endocytosis (Suresh et al., 2014). Generally, endocytic pathways, which is responsible for the uptake of nanomaterials, can be divided into three categories: the receptor-dependent endocytosis, the receptor-independent pathway and macropinocytosis (Pereira et al., 2015; Sevimli et al., 2015; Zhang et al., 2011). The receptor-mediated endocytosis is also known as clathrin-mediated endocytosis. The receptor-independent pathway can be further subclassified as caveolin-mediated endocytosis, clathrin-independent endocytosis, clathrin- and caveolin-independent endocytosis. Due to the fact that macropinocytosis is an endocytic pathway dominated by macrophages (Kerr and Teasdale, 2009; Lim and Gleeson, 2011; Swanson and Watts, 1995), current research was focused on the receptor-mediated and receptor-independent pathway. In this study, a diverse array of pharmacological endocytic pathway interference agents were used to ascertain the related intracellular track via nanoparticles for cell internalization (Ivanov, 2008). Chlorpromazine is a cationic amphipathic drug that blocks the clathrin-dependent uptake (Plummer and Manchester, 2013) Nystatin is a lipid raft blocker that specifically interferes with the caveolin-dependent endocytosis (Plummer and Manchester, 2013). Dynasore is a noncompetitive chemical inhibitor of dynamin activity and inhibits dynamin-mediated endocytic pathway and dynamin is related to several endocytosis pathways, including caveolin-dependent and clathrin-mediated pathways (Doherty and McMahon, 2009; Garcin and Panté, 2015; Plummer and Manchester, 2013). Firstly, to delineate whether clathrin- and caveolin-independent endocytosis is related to the internalization of the nanoparticles or not, dynasore is performed to validate the role of dynamin-mediated endocytic pathway. It is obvious that the dynasore leaded to a 45.3% and 48.4% reduction in the internalization of PEI and L25, respectively, which suggests that the clathrin-mediated endocytosis and caveolin-mediated endocytosis are probably involved in polyplexes internalization given that dynamin is a protein that is associated to both caveolin- and clathrin-mediated pathways (Fig. 8). Then, chloropromazine and nystatin were used to further elaborate the function of clathrin and caveolin in the endocytosis of the tested NPs. The cellular uptake data hints that the uptake amount of PEI and L25 with the addition of clathrin blocker chloropromazine was reduced by 50.7% and 46.8%, respectively, and no significant inhibition data was shown with nystatin treatments (Fig. 8). These results revealed clathrin-mediated pathway could be a domain route of PEI and L25 polyplexes that entry into the cells, whereas caveolin-dependent mechanisms were not favored. Taken together, our data indicate that the PEI and L25 likely follow the same internalization pathways and the clathrin-mediated endocytosis played a significant role in the internalization of PEG-Au PENPs and PEI.
Flow cytometry measurement of effect of the endocytic pathway inhibitors on the cellular uptake of PEI and L25. Chlorpromazine and dynasore demonstrated an approximately similar level of inhibition on the internalization of both nanoparticles. Nystatin almost not inhibits the cellular uptake of both nanoparticles. The control without inhibitors were normalized to 100% uptake (mean ± SD, n = 3).
4 Conclusion
In conclusion, PEG-Au PENPs synthesized using PEGylated PEI as a template with different Au atom/PEI molar ratios (25:1, 50:1, 100:1, and 200:1, respectively) were used as nonviral vectors for gene transfection and endocytosis studies. Cell viability experiment data displayed that all PEG-Au PENPs show much less cytotoxicity than PEI alone. Agarose gel retardation experiment and DLS assay illustrated that PEG-Au PENPs are able to compact pDNA and the formed polyplexes possess positive surface potentials and small sizes desirable for gene delivery applications. The PEG-Au PENPs complexed with two kinds of pDNAs encoding Luc and EGFP, respectively were examined for gene transfection studies. Our results clearly show that the entrapment of Au NPs within the PEGylated PEI do not dramatically compromise the gene transfection efficacy of PEI and show low-level cytotoxicity compared with PEI alone. The cellular uptake data indicated that the clathrin-mediated endocytosis is the main pathway in the internalization of formed nanomaterials. Therefore, the developed PEG-Au PENPs are more likely to be a great promising nanocarrier in various gene delivery applications.
5 Notes
The authors declare no competing financial interest.
Acknowledgments
This research is financially supported by the National Natural Science Foundation of China (31400816, 81761148028, 21773026), the Science and Technology Commission of Shanghai Municipality (17540712000), and the Fundamental Research Funds for the Central Universities (for X. Cao and X. Shi). X. Shi also thanks the support by FCT-Fundação para a Ciência e a Tecnologia (project no. PEst-OE/QUI/UI0674/2013, CQM, Portuguese Government funds) and by ARDITI-Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação, through the project no. M1420-01-0145-FEDER-000005-Centro de Química da Madeira-CQM+ (Madeira 14-20).
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