Article pubs.acs.org/Biomac
Albumin Incorporation in Polyethylenimine−DNA Polyplexes Influences Transfection Efficiency Marie-Isabel Syga,†,‡ Elena Nicolì,†,§ Esther Kohler,† and V. Prasad Shastri*,†,‡ †
Institute for Macromolecular Chemistry, University of Freiburg, 79104, Freiburg, Germany BIOSS−Centre for Biological Signalling Studies, University of Freiburg, 79104, Freiburg, Germany § Dipartimento di Scienze del Farmaco, University of Eastern Piedmont, 28100, Novara, Italy ‡
S Supporting Information *
ABSTRACT: Polyplexes of plasmid with synthetic polycationic vectors, such as linear polyethylenimine (LPEI), have been widely investigated. While much is known about the role of physicochemical characterization of the polycation in transfection, the role of serum components in the transfection using LPEI-polyplexes needs further investigation. In this study, bovine serum albumin was incorporated into the polyplex, either through precomplexation with circular DNA coding for green fluorescent protein prior to polyplex formation with LPEI or after formation of the polyplex. The transfection efficiency of these ternary polyplexes was then studied in HeLa cells. It was observed that the order of incorporation of albumin into polyplexes has a distinct effect on its uptake and transfection efficiency. Through colocalization and albumin depletion studies, we conclude that albumin plays a role in both the translocation of the complex into the cell and its unpackaging.
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INTRODUCTION Many diseases have their origins in genetic abnormalities, and these can be mitigated or reversed by introduction of healthy set of genes or silencing the defective gene. Some of the most promising candidates for gene therapy are severe combined immune deficiency (SCID), familial hypercholesterolemia, Duchene’s muscular dystrophy, and cystic fibrosis. The first example of successful application of gene delivery was reported by Anderson and co-workers, where girls suffering from adenosine deaminase (ADA) deficiency, an immune disorder, were treated by the injection of white blood cells that were transfected with the correct ADA gene, using retrovirus.1 Since then, several studies have been undertaken to reverse or stem the progression of diseases using gene therapy, a term introduced by Friedmann and Roblin in their landmark paper.2 A key aspect of gene delivery is the packaging of the genetic material into structures that can be efficiently transported into cells. In this regard, both viral and nonviral vectors have been extensively explored with varying degrees of success. Although viruses are excellent vehicles for the delivery of genetic material, they pose a few challenges and possess associated health risks.3−5 As a result, nonviral vectors such as synthetic polycations (PCs) have received much attention due to their perceived low immunogenicity and physicochemical diversity. PCs mediate the delivery of DNA by forming polyvalent (or multivalent) electrostatic complexes with the negatively charged DNA, termed polyplexes, which result in compaction of the DNA, making it permissive to cell entry. In this regard, © 2015 American Chemical Society
the efficient condensation of the DNA by the PC into a polyplex and its subsequent unpacking in the appropriate cellular compartment, while surviving the inhospitable environment of the endosome, remain the key steps that need further understanding.6 Among PCs, polyethylenimine (PEI) of 25 kDa molecular weight, first described by Boussif and coworkers in 19957 is considered the benchmark for in vitro transfections.8 Because PEI exhibits some degree of dosedependent cellular toxicity,8 several other PCs have been studied including modified PEI,9−11 polylysine,12 polyallylamine,13 poly-β-amino esters,14 and cationized albumin,15,16 which is albumin extensively chemically modified to bear positively charged moieties. While chemically modified albumin can be immunogenic, native albumin poses no such constraints. Several studies have shown that human albumin can enhance the transfection by lipoplexes.17,18 Carrabino and co-workers explored the effect of albumin in the delivery of PEI polyplex of plasmid encoding for luciferase in polarized lung epithelial cells lines A549 and 9HTE0, a human tracheal epithelial cell line.19 Again the incorporation of human albumin was shown to increase the transfection efficiency in these cells; however, no effort was made to systematically understand the effect of albumin concentration and mode of incorporation into the PEI−plasmid polyplexes on transfection. Interestingly, in all of Received: September 29, 2015 Revised: December 9, 2015 Published: December 11, 2015 200
DOI: 10.1021/acs.biomac.5b01308 Biomacromolecules 2016, 17, 200−207
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Biomacromolecules these studies, a role for albumin in the delivery of the lipoplexes into the cell was effectively ruled out. We had recently shown that albumin-coated siRNA-branched PEI (bPEI) polyplexes show enhanced silencing of eGFP expression in human endothelial cells. We further showed that the internalization of these albumin coated siRNA−bPEI complexes occurred to a great extent via caveolae.20 On the basis of these encouraging findings in this study, we have explored the ability of native unmodified albumin to modulate the physicochemical nature of the LPEI−plasmid polyplex. Specifically, we postulated a role for albumin as a molecular mediator of polyplex formation and complex dissociation, which are necessary for the release of DNA in the cytosol. Furthermore, we theorized that the transfection efficiency could be influenced by the order of incorporation of albumin in the DNA−LPEI polyplex (Scheme 1), which is a variable that has not been explored in past studies.
negatively charged phosphate group on the backbone of DNA) is required to obtain densely packed particles in order to get transfection without any free remaining DNA strands.7 Polyplexes were obtained by mixing the acidic LPEI solution, with acidic BSA solution premixed with pGFP, in cell medium according to Table 1. Below the isoelectric
Scheme 1. Schematic Representation of the Architecture of Albumin-Containing LPEI−Plasmid Ternary Complexes
point of BSA (4.7), the amino acids such as lysine, arginine, and histidine are protonated, thus conferring an overall positive charge to the albumin molecule, which is expected to promote interactions with pGFP. LPEI polyplexes without BSA were prepared in cell medium in absence of FBS at N/P ratios of 11, 22, and 33. Briefly, pGFP plasmid solution was prepared in cell culture medium (serum free, 0.0135 μg/ μL), and LPEI solution (1 mg/mL in H2O, pH 3) was added to the plasmid solution and incubated at room temperature for 10 min. Then it was diluted with FBS-free medium and was further incubated for 10 min to ensure polyplex formation. Polyplexes for light scattering, electron microscopy, and imaging were diluted in either H2O or 0.14 M NaCl solution instead of cell culture medium to avoid interference and artifact in the presence of serum proteins. For Type 1 (B-PX) polyplexes, BSA solution (1−100 μg/μL in 0.14 M NaCl solution, pH 3) was first added to the plasmid solution and incubated at room temperature for 10 min, following which LPEI solution was added and the system incubated for additional 10 min. Type 2 (PX-B) polyplexes were prepared using same procedure as the LPEI polyplexes followed by addition of BSA (Table 1). Characterization of polyplexes was performed using gel retardation assays, dynamic light scattering and transmission electron microscopy. Gel Retardation Assay. Complexes were diluted in 1X tris-borate EDTA buffer (TBE, Tris-base (calbiochem/Merck), boric acid (SigmaAldrich), ethylenediamine tetraacetic acid (EDTA) (Applichem, Darmstadt, Germany)) mixed with 1 μL of gel loading buffer (BlueJuice 10x, Life Technologies) and loaded in each lane (200 ng pGFP/10 μL/well) on a agarose gel (0.9% w/v, Thermo Fisher Scientific) in 1X TBE buffer. A 1Kb Plus DNA Ladder (Life Technologies) was used as a control for the verification of plasmid size. Analytical separation was performed in PerfectBlue gel electrophoresis chambers (PeqLab, Erlangen, Germany) using a protocol of 30 min at 90 V followed by 60 min at 150 V. Poststaining was performed in a solution of Nucleic Acid Staining GelRed (Biotium) for 30 min and DNA bands were visualized under UV using Fusion FX7 (Vilber, Eberhardzell, Germany). Dynamic Light Scattering (DLS) of Polyplexes. Size and zeta potential were measured with the Delsa Nano C particle analyzer (Beckman Coulter, California, U.S.A.) using the same sample for both techniques. Concentrated solutions of the polyplexes were analyzed in 1 cm polystyrene or quartz cuvettes and zeta potential measurements were performed in a flow cell provided by the manufacturer. The dilution of samples was performed with 0.14 M NaCl solution. Transmission Electron Microscopy (TEM) of Polyplexes. Polyplexes were prepared according to described protocol, diluted with ultrapure H2O and transferred to a copper TEM grid (CF-400Cu square mesh copper grid which was purchased from Electron Microscopy Sciences (Hatfield, U.S.A.)). Samples were negatively stained using 2 wt % uranyl formate solution and imaged using Zeiss LEO 912 Omega TEM at an accelerating voltage of 120 kV and the electron micrographs were analyzed with ImageJ.
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Table 1. Composition of Polyplexes Explored in This Studya particle formation code
particle type
I
II
III
PXz BXy By-PXz PXz-By
polyplex DNA−BSA Type 1 Type 2
DNA DNA DNA DNA
PEI BSA BSA PEI
medium 0% FBS medium 0% FBS PEI BSA
a
Transfection studies using of PXz, By-PXz and PXz-By were carried out in RPMI. y = μg BSA (20, 500, 1000, 1500, 2000 per 0.5 μg DNA). z = N/P ratios (11, 22, 33; μg PEI: 0.72, 1.40, 2.12 per 0.5 μg DNA).
MATERIALS AND METHODS
Materials. Linear polyethylenimine (LPEI, 25 kDa) was purchased from Polysciences Inc., (Eppelheim, Germany), bovine serum albumin (BSA) Fraction V was purchased from Calbiochem/Merck Millipore (Darmstadt, Germany), and sodium chloride was procured from Carl Roth (Karlsruhe, Germany). Lipofectamine2000 (Life Technologies, Darmstadt, Germany) was used according to the manufacturer’s protocol as a positive transfection control. Plasmid. pCMV-EGFP-C1 (4731 bp), coding for Enhanced Green Fluorescent Protein (EGFP), was kindly provided by BIOSS - Centre for Signaling Studies (University of Freiburg, Germany). The plasmid was replicated in a high-copying strain of DH5-α Escherichia coli in Luria−Bertani (LB) broth in the presence of kanamycin as the selecting antibiotic. pGFP plasmid was purified using GenElute Plasmid MaxiPrep Kit (Sigma-Aldrich, Munich, Germany), and the final concentration of plasmid was determined by UV absorbance at 260 nm using NanoDrop2000c (Thermo Fisher Scientific, Waltham, United States). Plasmid purity and size was analyzed using gel electrophoresis after enzymatic digestion (EcoR I and BamH I). Preparation of Polyplexes. For a given system, a minimum N/P ratio (ratio of positively charged nitrogen on the polymer molecule to 201
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Figure 1. Gel retardation assays for ternary albumin containing polyplexes and precursor complexes with 10 min formation time per mixing step. (A) LPEI−pGFP polyplexes PXz, Lane 1: DNA ladder; Lane 2: circular pGFP; Lane 3−12: PXz (N/P ratio z = 2.2, 4.5, 6.8, 9.1, 11.3, 13.6, 15.9, 18.1, 20.4, 22.6) and (B) pGFP−BSA (BXy) Lane 1: DNA ladder; Lane 2−3: circular pGFP; Lane 4−13: BXy (BSA/DNA ratio y = 20, 500, 1000, 1500, 2000 μg BSA/0.5 μg pGFP). Ternary albumin containing polyplexes with N/P ratio 22 are shown in (C) Type 1 (By-PX22); Lane 1: DNA ladder; Lane 2: pGFP; Lane 3−12: By-PX22 (BSA/DNA ratio y = 20, 500, 1000, 1500, 2000 μg BSA/0.5 μg pGFP) and (D) Type 2 (PX22-By) with same loading as (C). All gels were performed in TBE buffer, 0.9% agarose gel, 90−150 V. Cell Line and Cell Culture. Human cervical epithelial cancer cell line HeLa was provided genotyped by BIOSS toolbox (University of Freiburg) and cultured in RPMI 1640 (Roswell Park Memorial Institute, Life Technologies) supplemented with 10% FBS and 1% Penicillin−Streptomycin−Amphotericin (PAN-Biotech) and cultivated at 37 °C in a humidified incubator containing 5% CO2. Cell-Viability Assay. Cell viability was assessed 24 h after exposure to polyplexes using the standard MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) metabolic assay as per the manufacturer’s protocol. Metabolically active cells process MTT and convert into insoluble purple formazan crystals, which were then eluted using dimethyl sulfoxide and the absorbance of the solution was measured at 570 nm using Synergy HT microplate reader (BioTek, Winooski, U.S.A.). In Vitro Transfection. Cells (1.5 × 105 in 500 μL per well of cell medium with supplements) were plated into 24-well plates (Sarstedt, Nümbrecht, Germany) 24 h prior to transfection and were at 70−80% confluent at the time of transfection. All transfection experiments were carried out at least in triplicates. Before transfection, the medium was replaced by 500 μL of transfection medium with or without 10% FBS and the complexes were added to the cells at a final concentration of 0.5 μg DNA per well. Depending on the experimental conditions, the complete cell supernatant was replaced with fresh medium (10% FBS and antibiotics) after 4 h or until readout. Quantification of Transgene Expression (GFP). After 24 h of transfection, cells were prepared for analysis of transfection efficiency using flow cytometry. Thus, cells were washed with PBS (PANBiotech, Aidenbach, Germany), trypsinized, and resuspended in 500 μL of PBS solution containing 2% FBS. Flow cytometry was performed with BD LSRFortessa (BD Bioscience, Heidelberg, Germany) or with Gallios (Beckman Coulter) on FL2 channel (excitation laser: 488 nm; emission filter: 575/30 nm), and 5000 events were analyzed. Data analysis was performed with Flowing Software 2 (Perttu Terho). Cellular Uptake of Fluorescent-Labeled Polyplexes. Alexa Fluor 488 Protein Labeling Kit (Life Technologies) was used to label BSA. Alexa488-BSA was purified using 10 K centrifuge columns to remove unreacted dye. Cy3 Label It, Nucleic Acid Labeling Kit (Mirus Bio) was used to label the plasmid pEGFP-C1 with Cy3. Transfection was performed as described in 8-well chambers (Sarstedt), and after 24 h, HeLa cells were washed two times with PBS and fixed with 3.7% (v/ v) solution of formaldehyde (AppliChem) in PBS for 5−10 min. After subsequent washing the nucleus was stained by DAPI using Vectashield Mounting Media (Vector Laboratories, Burlingame, United States). Multichannel fluorescent images were acquired using
Observer Z1 (Zeiss) equipped with the Colibri LED imager using an oil immersion objective 63×. The images were analyzed with AxioVision (Carl Zeiss Microimaging, Release 4.8). Results and Discussion. Theory. Albumin is an amphoteric globular serum protein with an affinity to a notable amount of therapeutic drugs.21 As per our hypothesis, the introduction of albumin can lead to the evolution of two types of polyplexes as presented in Scheme 1. In the Type 1-polyplex albumin is added to DNA to generate a very weak polyvalent complex of BSA−DNA, and the subsequent addition of LPEI then results in the condensation of the BSA−DNA complex into a stable polyplex Type 1 (B-PX). In this architecture, the albumin is expected to play a mediatory role not only to facilitate intracellular unpackaging but also to stabilize the interactions of LPEI and DNA. In Type 2-polyplex, the albumin is added to previously prepared LPEI polyplexes to build a layer around the positively charged surface to obtain polyplex Type 2 (PX-B). This alternative polyplex architecture can promote cell uptake of DNA complexes through specific cellular interactions. In this study, the Type 1 and Type 2 polyplexes served as the basis to ascertain a role for albumin as a molecular glue (mediator) in the formation and unpackaging of LPEI-polyplexes. Agarose Gel Electrophoresis. The formation of LPEI polyplexes at N/P ratios beyond 9, particularly for ratios 11, 22, and 33, was confirmed using agarose gel electrophoresis (Figure 1). On the basis of this finding, the effect of introduction of albumin to the formation Type 1 and Type 2 polyplexes at N/P ratio of 11 and above was investigated. The condensation of the plasmid was evident in all N/P ratios, but in comparison to LPEI polyplexes, the formation of the polyplex was more robust at N/P of 22 and 33. In general all three classes of polyplexes (i.e., LPEI, Type 1 and Type 2) at all the N/P ratios showed complex formation immediately after mixing, with no noticeable loss in complexation even after 24 h, suggesting that the BSA−DNA−LPEI interaction occurs in a short time frame (Supporting Information Figure S1). We then investigated whether albumin alone was capable of condensing the plasmid. As expected, although the complexation of BSA to plasmid was verified by TEM (as discussed later), it appeared to be weak in nature, as dissociation of the BSA from the plasmid was observed under the conditions of normal gel electrophoresis of 90− 150 V bias. This was the case even when the pH of the BSA solution was reduced, and furthermore, even a gel run at a lower voltage of 25 V promoted dissociation of the BSA−plasmid complex. The presence of the free BSA band on the gel is expected and can be attributed to the excess of BSA used during complex formation. However, the gel retardation assays confirmed the formation of polyplexes under all 202
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from the average size of the poly aggregates with a mean size of 249 ± 35 nm, using DLS (Supporting Information Table S1). This discrepancy is explained by the fact that dynamic light scattering measures hydrodynamic radius and is more biased toward larger particles. All albumin-modified polyplexes showed low homogeneity and areas of free albumin with deformed polyplexes that made the results difficult to interpret. Thus, Type 1 polyplexes appeared on an average much larger compared to PX22 and had a fluffy appearance with particle mean size of 60.2 ± 23.5 nm (Figure 2C). In contrast, Type 2 polyplexes appeared to be an aggregation of smaller particles resembling a clover-like structure and had an average diameter of 44.1 ± 16.8 nm (Figure 2D). For comparison, the TEM of free albumin is clearly homogeneous with a mean size of 13.9 ± 3.1 nm (Figure 2E). The loose and uncompacted nature of Type 1 and Type 2 polyplexes is consistent with the trimodal distribution observed in light scattering. TEM images of Type 1 and Type 2 polyplexes have also shown the presence of uncomplexed albumin in the system, which is consistent with the gel retardation data. Transfection of Ternary Albumin Polyplexes. Transfection studies were undertaken using HeLa cells in the absence of serum. The transfection efficiency was assessed using flow cytometry. The ternary albumin polyplexes were compared to LPEI polyplexes at a constant N/P ratio of 22. Effect of Polyplex N/P Ratio on Cell Viability. The viability of HeLa cells after 4 h of incubation under all N/P ratios was determined in the absence and presence of serum in cell medium (Figure 3). In general, the cell viability was improved in the presence of serum. The reduction in cell viability induced by increasing LPEI during transfection is a known phenomenon that is well documented in literature.7,10 Furthermore, the addition of albumin had no adverse effect on the cell viability at all N/P ratios as also reported by previous studies.17,18 Therefore, the Type 1 and Type 2 polyplexes were deemed suitable for transfection. Transfection in Absence of Serum. As a reference, we observed that LPEI polyplexes transfected 16 ± 2% of the cell population in absence of serum in cell medium. As shown in Figure 4, Type 1 polyplexes showed dependency on albumin, and a maximum transfection of 22 ± 3% cells was observed at a BSA concentration
three scenarios LPEI, Type 1 and Type 2, which is a prerequisite for intracellular delivery. Dynamic Light Scattering. The size determination of ternary albumin containing polyplexes using light scattering resulted in trimodal size distribution (Table 2). A population with a diameter
Table 2. Size Distribution of Albumin Polyplexes As Determined by Dynamic Light Scattering of Type 1 Polyplexes of N/P Ratio of 22 and 50−400 μg BSA/20 μg DNA nm range
species
6−60 140−450 600−3000
BSA polyplexes aggregates of polyplexes
600 nm can be attributed to aggregates as shown later using TEM. Because large aggregates scatter light more efficiently, they overlap with the scattering intensities from the other populations, thus confounding the interpretation of the results. Therefore, polyplexes were additionally visualized using TEM. Transmission Electron Microscopy. First, to ascertain the impact of albumin in polyplex formation, we examined the DNA polyplex with only LPEI. TEM analysis was carried out with LPEI polyplexes of N/P ratio of 22, hereafter referred to as PX22, and they were used as a model system to understand the morphology and size of the polyplexes. Influence on polyplex size and structure caused by sample preparation in deionized water compared to ionic solutions was not further investigated, as the formation of sodium chloride crystals interfered with analysis of polyplex formation. Clear differences were detected among the three particle systems. PX22 polyplexes appeared highly uniform and showed no aggregation and had an average size of 30 ± 6 nm, as shown in Figure 2B. This value is significantly different
Figure 2. TEM images of LPEI polyplexes (black condensed spots), and both types of ternary albumin polyplexes with a fixed ratio of N/P 22 and B1000. Histograms were obtained using ImageJ with a normal distribution. 203
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Figure 3. Cell viability of Type 1 and 2 polyplexes in different cell medium during transfection (0% FBS or 10% FBS RPMI) was analyzed using MTT assay. Polyplexes were formed at N/P ratios 11, 22, and 33 with BSA concentration B20 and transfected in HeLa with incubation time of 4 h (B1000 and B2000 are shown in Supporting Information Figure S3). Assays were performed according to protocol after 24 h since polyplex addition.
Figure 4. Comparison of transfection efficiency of Type 1 and 2 polyplexes in HeLa cells quantified using flow cytometry. Cells were analyzed after exposure for 4 h in 0% FBS RPMI to polyplexes prepared at a N/P ratio 22 with increasing BSA concentrations.
Figure 5. Comparison of transfection efficiency of Type 1 and 2 polyplexes in HeLa cells quantified using flow cytometry. Cells were analyzed after exposure for 4 h in 10% FBS RPMI to polyplexes prepared at a N/P ratio 22 with increasing BSA concentrations.
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Biomacromolecules of 500 μg/well. Interestingly, a favorable regimen for transfection was observed between BSA concentration 20 μg and 1000 μg (per well), resulting in an increase of transfection efficiency. Beyond this, it is likely that more compact complexes are obtained with increasing albumin concentration, resulting in lesser transfection efficiency. Also, at higher albumin concentration, one can expect the complex to be less stable and undergo dissassembly, as the absence of albumin in the serum free transfection medium can potentially create a destabilizing osmotic gradient within the polyplexes. In contrast, in Type 2, where albumin is presumably located on the surface, no dependency on transfection efficiency was observed and transfection resulted in moderate mean value of 15 ± 5% transfected cells that is comparable to the intermediate Type 1 polyplex B1000. Transfection in HeLa Cells in the Presence of Serum. To explore the role of albumin in transfection by Type 1 and Type 2 in serumcontaining cell medium, the transfection was repeated in the presence of serum. Surprisingly, all Type 1 polyplexes showed significant loss in transfection efficiency with exception of B2000, which was comparable in presence and absence of serum (5 ± 1%), as shown in Figure 5. A reduction of transfection efficiency of LPEI polyplexes was also observed, which was decreased from 15% in absence of serum to 6% in the presence of serum. The maximum efficiency was obtained for Type 1 with 20 μg BSA with 12 ± 1% of the cells transfected. It is plausible that in the presence of excess albumin from the serum all complexes were enriched by albumin, and thus, the differences obtained in serumfree medium are normalized and/or diminished. This interesting observation gives circumspect evidence that in Type 1 polyplex albumin has a role in transfection. Likewise in the Type 2 complex, the presence of serum resulted in a loss of efficiency but only in the polyplexes with low albumin content (13 ± 1% → 6 ± 2%). This supports the contention that the excess albumin may cause reorganization of the polyplexes that affects its transfection potential. This, once again, points to a potential role for albumin in transporting the polyplex, as we have recently demonstrated in the delivery of siRNA and/or also a role in unpackaging of the complex, as discussed below.20 Influence of Albumin on Transfection Kinetics. In order to answer the question whether albumin just facilitates the interaction of the polyplexes with the cell membrane or if it also gets transported as part of the polyplex into the cell, we investigated the effect of albumin on the transfection. Therefore, the influence of free albumin or albumin supplemented in media versus albumin incorporated into LPEI polyplexes was studied in the following transfection experiment. The concentration of albumin in FBS supplement in cell medium (CBSA = 2.52 g/dL as per “Biochemical Prof ile and Hormone Prof ile of Fetal Bovine Serum” provided by Life Technologies) was used to establish the concentration of free albumin per well in 500 μL. Because FBS is supplemented at 10%v/v in media with 1% antibiotics, the concentration of albumin was determined to be 1135 μg per well. To ensure comparison polyplexes of Type 1 and 2 were made with 1135 μg of BSA and the transfection experiment was carried out in absence of serum, thus ensuring that the concentration of total albumin (free and in polyplex) was identical in all conditions. The polyplexes (Type 1, Type 2 and LPEI) were incubated with HeLa cells for 4 and 24 h. Although all cells received the same numerical amount of all components: albumin, LPEI and pGFP plasmid, the Type 1 and Type 2 polyplexes showed different transfections compared to LPEI polyplexes at 4 and 24 h (Figure 6). After 4 h of incubation with the albumin-mediated transfection was 2.5-fold higher than that by LPEI-polyplexes by itself. This suggests that albumin accelerates the transfections process. However, interestingly after 24 h the transfection efficiency was higher in the LPEI polyplexes versus the Type 1 and Type 2 polyplexes. This provides further circumspect evidence for a role for albumin in the transfection process, as the reduced transfection observed in the albumin containing polyplexes may be attributed to the exocytosis or the albumin−LPEI−plasmid complex. It is well-known that albumin can undergo transcytosis22 across endothelial23 and epithelial cells membrane24 and undergo transcytosis when they engage the Gp6023,25 receptor that resides in the caveolae. Because we have shown in an
Figure 6. Flow cytometry of GFP expression in HeLa cells induced by albumin-modified polyplexes Type 1 and Type 2 in serum free RPMI medium compared to LPEI−pGFP polyplexes (N/P ratio 22) in RPMI medium (10% FBS). earlier study that siRNA−bPEI complexes containing albumin are internalized to a great extent by the caveolae-mediated endocytosis, this explanation is rather consistent with that finding.20 It is also clear that association of the albumin with the polyplex is necessary, as the presence of free albumin alone did not accelerate the transfection in the case of LPEI polyplexes (Figure 6). Role of Albumin during Polyplex Internalization. Having demonstrated that albumin plays a role in transfection, we further investigated if the faster rate of transfection is due to improved interaction of the polyplex with the cell membrane or due to additional albumin being internalized as part of the polyplex into the cytosol, thereby potentially playing a role in unpackaging of the polyplex. Polyplexes were formed using Cy3-labeled pGFP, and their fate was followed in HeLa cells using fluorescence microscopy. For comparison, transfection was carried out in medium with or without serum. Internalization of Cy3-pGFP for all three polyplexes showed no bias with respect to uptake (Figure 7). We have shown in an earlier study that albumin mediates the uptake of siRNA−bPEI polyplexes into endothelial and epithelial cells and that this uptake of smaller oligonucleotides like siRNA is mediated via caveolae.20 Although the
Figure 7. Fluorescent micrographs of Cy3-labeled pGFP and DAPIstained nuclei of HeLa cells were taken 4 h after transfection. Transfections with the three types of polyplexes were performed in the absence and presence of serum. Type 1 and Type 2 polyplexes were prepared at a N/P 22 and with a BSA concentration of 20 μg (B20). 205
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Figure 8. Co-localization analyzed with fluorescent microscopy of labeled Type 1 polyplexes B20‑PX22 in serum-free RPMI medium after 4 h of uptake including Alexa488-labeled BSA. Confocal microscopy imaging revealed localization close to the nucleus inside the cell, excluding attachment onto the cell surface. albumin layer in Type 2 can prevent binding of other serum components that can interfere with the cellular uptake (Figure 5). According to the theory of dissociation behavior described in Scheme 2, in Type 2 polyplexes, the albumin covers mainly the surface
size of pGFP plasmid (∼3000 kDa) is significantly larger than siRNA (∼16.5 kDa) the uptake mechanism might be similar but needs to be further investigated. After verifying that pGFP plasmid was translocated into the cells in both Type 1 and Type 2 polyplexes, a colocalization study was undertaken using the Type 1 particle, as this is the polyplex where albumin, being in the intermediary layer, is most likely to influence the dissociation behavior of the polyplex. Because frequent colocalization of Alexa488-labeled BSA with Cy3-labeled pGFP was observed along with the presence of uncomplexed albumin (Figure 8), the polyplex can be assumed to have transported into the cell as an intact ternary complex. Thus, the dissociation of BSA out of the complex prior to internalization can be considered to be highly unlikely. The presence of free BSA inside the cell is most likely a combination of the uptake of uncomplexed BSA and the dissociation of the polyplex inside the cell during transfection time. For comparison, the uptake behavior of albumin itself in HeLa is shown in Supporting Information Figure S4, and it is clear that exogenous albumin, when taken up by HeLa cells, was primarily localized in the perinuclear region. However, no such localization was observed in the uptake of Type 1 polyplexes, suggesting that this was caused by the dissociation of the complex. As confirmed by quantifying gene expression, the internalization of Type 1 particles into the nucleus in serum containing medium was low. During endocytic uptake of gene vehicles into the acidic endosome, PEI is thought to buffer the hydrogen ions due to its partially protonated amino groups and thus inducing an escape by swelling and bursting of the endosomal compartment.8,26 On the basis of our findings, we postulate that especially in Type 1 formation that the albumin in the core plays an additional role for endosomal escape by providing an osmotic effect to facilitate the unpackaging of plasmid thereafter. We have shown that albumin and plasmid form a loose complex (Figure 1B) (part of formation of Type 1) and that this is an advantage rather than a disadvantage. The release of plasmid out of this loose Type 1-complexation is less cumbersome than relieving the plasmid out of strong multivalent electrostatic interaction with cationic LPEI in Type 2 polyplexes. Thus, the very factor that supports strong polyplex formation also prevents its unpackaging inside the cell. This promotes our hypothesis of optimized packaging by loosely formed complexes (as in Type 1) that also maintain the ability to release the plasmid rapidly in contrast to the strong condensation that occurs with LPEI alone. Reasons for such observations are explained with a theory hypothesized by Lu and co-workers27 that describes the role of albumin-coating in lipid-based nanoparticles during siRNA delivery. In that study, it was theorized that albumin absorbed on the surface of particles, prevented the specific adsorption of abundant large proteins contained in serum (e.g., immunoglobins) that reduced the internalization efficiency. Extension of that finding to our observations suggests that additionally in the case of Type 2 complexes, the presence of an albumin coating could enhance the uptake of the polyplex by mitigating interference by serum. This aspect could enable Type 2 polyplexes to have a transfection efficiency higher than LPEI polyplexes in serum containing medium condition, because the
Scheme 2. Dissociation Process Hypothesized for Albumin Polyplexes Type 1 and Type 2
and therefore exchanges with the environment, showing a transfection pattern similar to polyplexes prepared without albumin. Taken in sum, the findings of this study show that serum components, such as albumin, can play a useful role in mediating transfection using synthetic polymer vectors.
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CONCLUSIONS In this study, we theorized that by using a ternary system involving macromolecular species that have electrostatic affinity for DNA, and polycation, the association and dissociation of polyplexes may be controlled. Using albumin as a model macromolecule, the characteristics of polyplex were modified during their formation. We have shown that transfection efficiency is dependent not only on the components but also on the architecture of the packaging system that transports the plasmid into the cell. Comparison of Type 1 and Type 2 polyplexes revealed that besides the order of mixing, the introduction of albumin played a significant role in polyplex dissociation and transfection rate as theorized previously. Additionally, we have demonstrated that the transfections can be more efficient when albumin forms the intermediary layer between the pGFP and LPEI, as it is the case in the Type 1 polyplex, where accelerated and improved transfection efficiency is observed under serum-free conditions. 206
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01308. Supplementary figures and table (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
V.P.S. and M.S. conceived the project. M.S., E.N., and V.P.S. designed the experiments. M.S., E.N., and E.K. carried out the experiments. M.S., E.N., E.K., and V.P.S. wrote the manuscript. All authors have approved the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors wish to thank Rolf Thomann for assistance with transmission electron microscopy, Pavel Salavei for assistance with flow cytometry, and Jon Christensen and Julia Voigt for valuable discussions. The authors wish to acknowledge the excellence initiative of the German Federal and State Governments Grant EXC 294 (Centre for Biological Signalling Studies) and the University of Freiburg for funding this effort.
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DOI: 10.1021/acs.biomac.5b01308 Biomacromolecules 2016, 17, 200−207
