Intracellular Availability of pDNA and mRNA after Transfection: A

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Intra-cellular availability of pDNA and mRNA after transfection: A comparative study in between polyplexes, lipoplexes and lipopolyplexes Cristine Gonçalves, Sohail Akhter, Chantal Pichon, and Patrick Midoux Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00376 • Publication Date (Web): 03 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Molecular Pharmaceutics

Intra-cellular availability of pDNA and mRNA after transfection: A comparative study in between polyplexes, lipoplexes and lipopolyplexes

Cristine Gonçalves1, Sohail Akhter1,2, Chantal Pichon1 and Patrick Midoux1*

1

Centre de Biophysique Moléculaire, CNRS UPR4301, rue Charles Sadron CS 80054 F-

45071 Orléans Cedex 02 and Université d’Orléans, France Phone: + 33 2 38 25 55 65; 2

Le Studium® Loire Valley Institute for Advanced Studies, Centre-Val de Loire région,

France

[*]Corresponding-Author: [email protected]

Keywords: Gene transfer; mRNA transfer; Cationic lipids; Cationic polymers; Cytosolic delivery; endosome escape.

1 ACS Paragon Plus Environment


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Abstract Intracellular availability of nucleic acids from synthetic vectors is critical and directly influences the transfection efficiency (TE). Herein, we evaluated the TE of polymer- and lipid- based nanopolyplexes (polyplexes, lipoplexes and lipopolyplexes) of EGFP-encoding mRNA and pDNA. To determine the translation and transcription efficiency of each nucleic acid nanoplex, in vitro expression was measured in HEK293T7 cells that permit gene expression in the cytoplasmic region. Globally, mRNA transfection profile was well corroborative with cytoplasmic transfection of pT7-pDNA as well as with nuclear transfection of pCMV-DNA. Irrespective of the nucleic acid, high TE was observed with Histidinylated lpolyethyleneimine (His-lPEI) polyplexes and dioleyl succinyl paromomycin/O,O-dioleyl-Nhistamine phosphoramidate (DOPS/MM27) lipoplexes. Moreover, His-lPEI polyplexes yielded higher in-vitro expression of EGFP for pDNA than for mRNA. Furthermore, a significant enhancement in the TE in the presence of an excess of His-lPEI was observed indicating that this polymer promotes cytosolic delivery. Compared to other nanoplexes, HislPEI polyplex showed high intracellular availability of DNA- and mRNA along with low cytotoxicity, owing to its rapid (complete or partial) unpacking in the cytosol and/or endosomes. This study gives an insight that, whether with mRNA or pDNA, enhancing nanoplex unpacking in the endosomes and cytosol would improve the delivery of nucleic acid in the cytosol and particularly in the case of pDNA where a sufficient available amount of pDNA in the cytoplasm would definitely improve its transport towards the nucleus.


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Introduction Non-immunogenicity, relatively non-toxic nature and the potential of targeting to specific cells are increasingly making the synthetic vectors the carriers of choice for DNA and mRNA delivery for various therapeutic purposes including gene therapy. These synthetic vectors are lipid-, polymer- or combination of lipid-polymer-based and accordingly called as lipoplexes, polyplexes or lipopolyplexes, respectively.1-4 However, these synthetic vectors experience inferior transfection efficiency (TE) compared to the viral vectors.2,4 Therefore, optimizing an efficient and safe synthetic vector for DNA and mRNA cellular delivery remains a major challenge to their successful clinical translation. A novel synthetic vector should be able to provide protection of the nucleic acid payload and promote endosomal escape,5-7the unpacking of complex and the release the nucleic acid in the cytosol. To improve the transfection ability of nanoplexes, recently lipids, polymers, and cyclodextrins containing ethyleneimine

8-15

and/or histidine moieties

16-19

were investigated

and proved to improve the endosomal escape by exploiting the protonation of their secondary amines or imidazole groups. In the case of pDNA, its nuclear delivery is favoured when it is inserted with a specific sequence that can be recognized by transcription factors such as NFκB that acts as shuttle between the cytosol and the nucleus.20-23 Furthermore, the pDNA transfection efficacy can be improved by linking pDNA to a specific microtubule-peptide that allows pDNA to migrate towards the nuclear envelope.24 However, given complexity of such design, the use pDNA in the nanoplexes is more preferable. It should be stressed that the nuclear delivery of pDNA is highly dependent on its availability in the cytosol which ultimately depends on the efficiency of endosomal escape and the dissociation of pDNA nanoplexes. Likewise pDNA, the transfection of synthetic mRNA is an attractive strategy that has been extensively used in recent years for therapeutic vaccination against cancer and viral diseases.25-28 mRNA accessibility in the cytosol by the transcription machinery depends on



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the endosomal escape and the dissociation of mRNA complexes, as for pDNA transfection yet without the need of nuclear transport. There are very limited reports on designing versatile nanoplexes based on reducible cationic lipids, polymers and combination of both that facilitate efficient and stable cytosolic availability of pDNA and mRNA. Herein, we investigated the cytosolic availability of pDNA and mRNA and eventually its impact on the TE of the polyplexes, lipoplexes and lipopolyplexes in HEK293T7 cells. This cell line is an excellent tool to assess the endosomal escape efficiency and the cytosolic accessibility of mRNA and pDNA. These cells steadily express T7 RNA polymerase that allows the cytoplasmic transcription of genes under the control of the bacteriophage T7 RNA polymerase promoter. Moreover, the cytoplasmic availability of pDNA and mRNA from the polyplexes, lipoplexes and lipopolyplexes were compared with regards to the in vitro translation/transcription machinery. Materials and methods Reagents All reagents were purchased from Sigma (St. Quentin Fallavier, France) unless otherwise stated. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (5,000 IU/mL penicillin and 5,000 µg/mL streptomycin), and phosphate-buffered saline (PBS) were supplied by PAA Laboratories (Les Mureaux, France). O,O-dioleyl-phosphoramidate arsonium (KLN47), O,O-dioleyl-N-(3N-(N-methylimidazolium iodide)propylene) phosphoramidate (KLN25) and O,O-dioleyl-N-histamine phosphoramidate (MM27) were kindly given by Prof. P-A. Jaffrès and Dr. M. Berchel (CEMCA, CNRS UMR 6521, IFR148 ScInBioS, UBO, Brest, France).29-32 Dioleyl succinyl paromomycin (DOSP; exhibits 4 cationic charges per molecule at pH 7.4) was kindly given by Drs. B. Pitard and M. Mevel (INSERM, U915, Nantes, France).33 KLN47, KLN47/MM27 (1/1; mol:mol), KLN25/MM27 (1/1; mol:mol), DOSP/MM27 (1/1; mol:mol) liposomes were prepared by the


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film hydration method.34 Lipofectamine2000™ (LFA) comprising DOSPA/DOPE (3/1; mol:mol) was procured from Invitrogen (Pontoise, France). Histidylated poly-L-Lysine (HpK), a polylysine of 190 lysine residues substituted with 95 histidine groups and its derivative PEGylated histidylated poly-L-Lysine (PEG-HpK) grafted with one mPEG 5kDa molecule

per

molecule

were

synthesized

as

previously

described.18,

35

Linear

polyethylenimine (lPEI; Mw 22kDa) was kindly given by Prof P. Guégan (UPMC UMR 7610, Ivry sur Seine, France). His-lPEI (lPEI modified with 16% histidine residues per molecule) was purchased from Polytheragene (Genepole, Evry, France).17 At pH 7.4, PEGHpK, lPEI and His-lPEI exhibited 190, 102 and 85 cationic charges per polymer molecules, respectively17. Plasmid DNA pCMV-luc (pTG11033, 9514 bp, Trangène S.A., Strasbourg, France) and pT7-Luc (pGEM4Z-Luc-A64, 4405 bp)35 were encoding the Photinus pyralis firefly luciferase (Luc) gene under the control of human cytomegalovirus (CMV) and the bacteriophage T7 RNA polymerase promoter, respectively. pCMV-EGFP (5130 bp) and pT7-EGFP (pGEM4ZEGFP-A64, 3373 bp)

35

were homemade plasmid DNA encoding the jellyfish Aequorea

victoria enhanced green fluorescent protein (EGFP) under the control of the human cytomegalovirus (CMV) and the bacteriophage T7 RNA polymerase promoter, respectively. Supercoiled DNA was isolated from E. Coli DH5α supercompetent (Invitrogen, Cergy Pontoise, France) by alkali lysis and purification with QIAGEN Mega Kit Endotoxin-free Plasmid (Qiagen, Courtaboeuf, France). pCMV-Luc was labelled with fluorescein using the Label IT nucleic acid labelling kit (MIRUS, Madison, WI, USA) at 1:2 reagent/pDNA weight ratio.34 In vitro transcribed mRNA



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pGEM4Z-EGFP-A64 and pGEM-4Z-Luc4 plasmids were linearized with SpeI. Linearized plasmids were used as template for in vitro transcription using the mMessage mMachine T7 Transcription Kit(Ambion, Oxfordshire, United Kingdom) to produce antireverse cap analog (ARCA)-A100 mRNAs as described elsewhere.35 Synthetic mRNA-EGFP and mRNA-Luc had an ARCA modified cap and a 100-adenosine poly(A) tail. The RNA concentration was determined by measuring the absorbance at 260 nm; RNA had 260:280 ratios ≥2 and was stored at -80°C in small aliquots. Cells and Cell Culture Human embryo kidney 293T7 cells were cultured in DMEM (containing 10% FBS, 2 mM L-glutamine, 1mM sodium pyruvate, 100 units/mL penicillin, 100 units/mL streptomycin, and 400 µg/mL geneticin).36 Cells were incubated at 37°C in a humidified atmosphere (5% CO2). Mycoplasma-free condition of cells was evidenced by the MycoAlert® Mycoplasma Detection Kit (Lonza, Levallois Perret, France). Polyplexes, Lipoplexes and Lipopolyplexes The polyplexes were prepared by mixing by up-down pipetting of HpK, PEG-HispLK, His-lPEI or lPEI in 10 mM HEPES buffer (pH 7.4) to pDNA (7.5 µg) in 10 mM HEPES buffer (pH 7.4) at different weight ratio followed by 4 sec of vortex mixing. The solution was kept for 30 min at room temperature before use.34 Likewise, lipoplexes were prepared by adding pDNA (7.5 µg) to the liposomes in 10 mM HEPES buffer,(pH 7.4) and the solution was kept for 30 min at room temperature before use.34 LPD100 and LPD16 lipopolyplexes were prepared by adding liposomes to polyplexes.37 In brief, firstly PEG-HpK or His-lPEI (15 µg in 15 µL 10 mM Hepes buffer, pH 7.4) were added to pDNA (7.5 µg in 97 µL 10 mM HEPES buffer, pH 7.4) with up-down pipetting to make polyplexes. This solution was mixed for 4 sec with a vortex mixer and kept for 30 min at 20°C. Finally, KLN25/MM27 liposomes (9 µL at 5.4 mM in 290 µL 10 mM HEPES buffer, pH 7.4; 10 µg) were added to the


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polyplexes by up-down pipetting. The solution was kept for 15 min at 20°C before use. For transfection, the polyplexes, lipoplexes and lipopolyplexes solutions were adjusted to 1.5 mL with serum-free medium. The control formulation, LFA lipoplexes, was prepared according to the manufacturer’s instructions using a DNA/lipids weight ratio of 1/2. The synthetic mRNA polyplexes, lipoplexes and lipopolyplexes were prepared in a similar fashion as presented above for pDNA, Particle size distribution and ζ potential measurements The particle size of DNA complexes was measured via dynamic light scattering recorded at 90° angle to incident radiation using SZ-100 Analyser (Horiba Scientific, les Ulis, France). For the analysis, the samples were prepared in cuvettes by adding 50 µl of solution with DNA complexes into 1.45 mL of 10 mM HEPES buffer (pH 7.4). The particle size as hydrodynamic diameters were calculated from the size distribution by volume (generated by the Stokes-Einstein equation for polydisperse samples), provided by the inbuilt software, and are reported as the average of 3 independent measurements ± the deviation from the mean. The uniformity of size distribution was recorded as polydispersity index (PDI) obtained with the particle size. The ζ potential measurement (measured by electrophoretic mobility) was also recorded on the SZ-100 Analyser using the Zeta mode to monitor the global surface charge of DNA complexes. Cells transfection and EGFP expression Two days prior to transfection, HEK293T7 cells were seeded at 5 x 105 cells per well in 1 mL of culture medium in a 24-well plate. The nanoplexes (500 µL; 2.5 µg DNA and 5 µg mRNA in their respective transfection) were incubated with the cells for 4 h at 37 °C. Afterwards, the medium was replaced with completely fresh medium. Cells were washed twice with PBS (pH 7.4), harvested with trypsin, centrifuged (1500 rpm for 5 min at 4°C) and suspended in PBS. EGFP expression was quantified 48 h post-transfection by flow cytometry



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(LSR, Becton Dickinson) by measuring the cell-associated fluorescence intensity at λex = 488 nm and λem = 520 ± 24 nm). Cytotoxicity 48 h after transfection, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 100 µL of 5 mg/mL solution in PBS) was added to each well, and the cells were incubated for 4 h at 37°C. The MTT converted to Formosan was solubilised with acidic isopropanol and quantified by measuring the absorbance at 570 nm with spectrophotometer. The data are presented as the mean ± SD of two experiments, each performed in triplicate and they are expressed as the percentage of the absorbance of non-treated cells. In vitro transcription and translation In vitro (cell-free) transcription and translation experiments were performed using TNT Quick Coupled Transcription/Translation Systems Kit (Promega, Charbonnières-lesBains, France) following the manufacturer instructions. In a 1.5 mL tube, 0.2 µg of pT7-Luc plasmid or synthetic mRNA-Luc in 2.6 µL HEPES buffer (10mM, pH 7.4) free or complexed with nanoplexes at various N/P charge ratios were mixed with 40 µL of Quick Master Mix and 1 µL of Methionine (1 mM). The volume was adjusted to 50 µL with miliQ water and the mixture was incubated for 1 h 30 min at 30°C. Finally, 0.5 µL of this mixture was added to 50 µL of LAR (Luciferase Assay Reagent) and the luciferase activity was measured two times for 10 sec with a 2 sec interval using a luminometer (LUMAT LB 9507, Berthold Technologies, Bad Wildbad, DE). Dye exclusion assay Ethidium bromide (EtBr; 14 µM) was added to pDNA (1 µg) in 0.7 mL HEPES buffer (10 mM, pH 7.4) to obtain a pDNA/EtBr solution. Then aliquots of synthetic vector (made in 10 mM HEPES buffer, pH 7.4) were added to the pDNA/EtBr solution and the fluorescence intensity (λ ex = 530 nm; λem = 580 nm), which is the representative of the EtBr exclusion,


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was measured. The percentage of pDNA complexed was accordingly evaluated as: (Fsample Fmin)/(FDNA - Fmin) where Fsample , FDNA, and Fmin are the fluorescence intensities of EtBr in the presence of DNA plus synthetic vector and in the presence of DNA alone and the maximum of the fluorescence quenching of EtBr, respectively. Plasmid DNA Uptake Cells were incubated at 37°C for 4 h with different DNA nanoplexes prepared with fluorescein-labelled pDNA. Then, cells were washed twice with PBS harvested with trypsin, centrifuged (1500 rpm for 5 min at 4°C) and suspended in PBS. The cell-associated fluorescence intensity was measured by flow cytometry at λex = 488 nm; λem = 520 ± 24 nm before and after the post-treatment with monensin in order to evidence the presence of fluorescein-labelled pDNA in acidic or neutral environment.38 Statistics Results are expressed as the mean ± standard deviation. The mean of each group was compared by an analysis of variance (ANOVA) followed by a t Student test. p < 0.05 was considered as statistically significant. Results The nanoplexes of pDNA and mRNA were prepared at different N/P ratio according to the weight ratio of nucleic acid to the polymers and/or lipids that gave complete complexation. There were no major difference in the particles size distribution and surface charges of the nanoplexes while comparing the DNA and mRNA nanoplexes (Table 1). The transfection and expression of protein studies were conducted in HEK293T7 cells allowing direct comparison of the TE for a gene expressed in the nucleus (pCMV-DNA) to gene expressed in the cytosol (pT7-DNA) and mRNA translation in the cytosol (Fig. 1). Transfections of pDNA and mRNA were performed with (i) polyplexes prepared either with lPEI, His-lPEI or PEG-His-pLK, (ii) lipoplexes made up of cationic liposomes comprising



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cationic lipophosphoramidates - bearing either an arsonium (KLN47/MM27) or an imidazolium (KLN25/MM27) cationic head group - or with a dioleyl succinyl paromomycin derivative which is characterized by an aminoglycosidic group (DOSP/MM27) (Fig. S1).34 Those cationic lipids were associated with a lipophosphoramidate bearing an imidazole polar head as a pH-sensitive co-lipid (MM27). Lipopolyplexes LPD100 and LPD16 were prepared by adding KLN25/MM27 liposomes to polyplexes made either with PEG-His-pLK or HislPEI.34 The distinctive feature of all these vectors is the presence of the amines and imidazole groups that favour the endosome destabilization in acidic medium. pDNA transfection in the nuclear expression system We compared the TE in HEK293T7 cells by transfecting with pCMV-EGFP nanoplexes. For each type of DNA complex, the charge ratio (N/P) used in this study was kept the same as previously optimized by us in the transfection of C2C12 cells.34 Figure 2A shows the percentages of EGFP-positive cells, the mean fluorescence intensities (MFI) of the cells and cytotoxicity. The MFI are expressed relative to a randomly chosen MFI value (2408) of His-lPEI complexes. Figure 2A shows the percentages of EGFP-positive cells, the mean fluorescence intensities (MFI) of the cells and cytotoxicity. The MFI are normalized to the MFI value of His-lPEI complexes, considered as 100%. His-lPEI and DOSP/MM27 appeared as the best vectors with about 60% transfected cells and highest MFI. On the other hand, among these two formulations, His-lPEI induced less cytotoxicity (10%) than DOSP/MM27 (20%). The percentage of EGFP-positive cells and MFI were relatively lower (p < 0.05) with LFA, lPEI, KLN25/MM27, LPD100 and LPD16 with not much difference among them. Very poor TE with PEG-HpK polyplex resulted from its low uptake due to the presence of long chain PEG that eventually prevents its electrostatic interaction with the cells. pDNA transfection in the cytoplasmic expression system


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Figure 2B displays the overall TE as percentage of transfected cells and MFI measured in HEK293T7 cells after transfection with the pT7-EGFP plasmid complexed with the vectors investigated in this study. Transfections with the pT7-EGFP-based nanoplexes were performed at the same N/P charge ratios stated elsewhere in the paper. As the results indicate, His-lPEI and DOSP/MM27 based complexes have again emerged as the best in terms of the number of transfected cells and MFI. However, among these two complexes, the MFI of HislPEI complex was higher (p < 0.05). Conversely, lPEI, due to its high cytotoxicity, ends up less efficient. Nanoplexes of KLN25/MM27, LPD100 and LPD16 led to a moderate transfection. Figure 3 shows the global TE (i.e. the number of EGFP-positive cells × MFI) of two gene expressing systems (pCMV-EGFP and pT7-EGFP) indicating a good correlation between the nuclear-dependent (pCMV-EGFP) and the nuclear-independent (pT7-EGFP) transfections. Of note, the TE level with pT7-EGFP is ~10-fold lower than the one achieved with pCMV-EGFP due to theCMV promoter that drives strong gene expression of EGFP. mRNA transfection To evaluate the versatility of cationic polymers and liposomes, we compared the transfection of their complexes with mRNA-EGFP. The mRNA-EGFP produced by in vitro transcription was end-capped in 3’ and 5’ by the anti-reverse cap analogue ARCA and Poly(A) tail of 100 adenosines, respectively. This mRNA construct is 700-fold better efficient than the m75’Gppp5’G capped mRNA with a Poly(A) tail of 64 adenosines.39 The delivery of mRNA using His-lPEI, KLN25/MM27, DOSP/MM27 and LFA allowed transfection of more than 50% of cells (Fig. 4A). The N/P values of these mRNA complexes with lPEI, KLN25/MM27, DOSP/MM27 and LFA were 1.5, 1.8, 4 and 3.3, respectively. Due to low level of transfection with 2.5 µg mRNA and also to cytotoxicity concerns of some vectors with 5µg mRNA at the same N/P values used with pDNA complexes, the transfections were nevertheless carried out with 5 µg mRNA but the N/P values were kept lower (Fig. S2.1-



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S2.7). As expected, the transfection control (LFA) showed higher EGFP-positive cells and significantly high EGFP expression, as indicated by the MFI results (Fig. 4B). The highest TE, as determined from the percentage of EGFP-positive cells and EGFP expression (MFI), was obtained with His-lPEI and LFA (Fig. 4C). His-lPEI polyplexes exceptionally allowed high mRNA transfection along with low cytotoxicity at N/P ratio of 5. Though, the number of EGFP-positive cells was high with KLN25/MM27 and DOSP/MM27 based complexes, the EGFP expression in these cells is interestingly 5 to 6-fold lower when compared to the HislPEI and LFA based complexes. Compared to pT7-EGFP transfection, mRNA-EGFP transfection produced 10% to 20% higher number of EGFP-positive cells with His-lPEI, KLN25/MM27, DOSP/MM27 and LFA nanoplexes (Fig. 5A). mRNA transfection with LPR100 led to a lower number of EGFPpositive cells than LPD100-DNA transfection and a similar trend was observed with lPEI lipopolyplexes,LPR16 and LPD16. EGFP expression was always higher after transfection with pT7-EGFP and produces more uncapped mRNA-EGFP without polyA tail than capped mRNA-EGFP bearing a long polyA tail. Predictably, the number of mRNA-EGFP transcripts is greater after transfection with pT7-EGFP than mRNA-EGFP transfection (Fig. 5B). Finally, looking at the global data, the mRNA transfection profile in terms of vectors is well corroborative with the pT7-DNA transfection (Fig. 5C). pDNA condensation The strength of the interaction between pDNA and some vectors was assessed by dye exclusion experiments using EtBr (Fig. 6). The fluorescence quenching maxima obtained with the pDNA complexed with pLK, lPEI, His-lPEI, DOSP/MM27 and KLN25/MM27 was 100%, 90%, 80%, 70% and 65%, respectively. The dye exclusion depends on the pDNA condensation capacity of the cationic vector. The fluorescence quenching maxima directly reflect the pDNA condensation levels in the complexes. Fig. 6 clearly indicates the decreasing


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order of pDNA condensation rate: pLK ≈ lPEI > His-lPEI > DOSP/MM27 > KLN25/MM27. This result was further confirmed by agarose gel electrophoresis shift assay showing that all pDNA was complexed with polycations at N/P ratio > 2 (Fig. S2.1 and Fig. S2.2). In contrast, free pDNA was visible till N/P ratio of 4 with KLN25/MM27 and DOSP/MM27 liposomes (Fig. S2.3 and S2.4). pDNA accessibility by the transcriptional machinery Next, in vitro transcription/translation of pT7-Luc was performed as a function of N/P ratio to assess the pDNA accessibility within the complexes. For this purpose, the luciferase activities produced by pT7-Luc nanoplexes were measured after incubation with reticulocyte extracts (Fig. 7). It was found that except for His-lPEI complex (luciferase activity: 40% at N/P ratio of 6), the luciferase activity dropped sharply in the case of cationic polymer complexes when the N/P ratio was higher than 1 or 2. Moreover, the luciferase activity dropped when the amount of cationic liposomes (DOSP/MM27 and KLN25/MM27) was increased in the complexes. Overall, the transcriptional activity drop was in correlation with the pDNA complexation level (Fig. 7 ▲). Based on the results of the dye exclusion experiments and agarose gel electrophoresis, the observed luciferase activity with pT7-Luc lipoplexes was ascribed to the amount of non-complexed pT7-Luc. Therefore, no transcription/translation occurred when pT7-Luc was strongly complexed with the vectors. In contrast, the transcription/translation machinery was not drastically impeded with HislPEI/pDNA complexes. Accordingly, pT7-Luc complexes made with lPEI, KLN25/MM27 or DOSP/MM27 at the N/P ratios used to transfect HEK293T7 cells did not allow in vitro transcription/translation. However, the result was otherwise with His-lPEI polyplexes; in the case of the transfection of HEK293T7 cells, the observed EGFP expression with pT7-EGFP strongly suggests that complete or partial dissociation of pT7-EGFP from polyplexes or lipoplexes occurred. We anticipated that it was also the case for pT7-Luc complexed with



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PEG-His-pLK, KLN47, KLN47/MM27 and LFA. The transcriptional activity of pT7-Luc in lipopolyplexes-LPD100 and LPD16 made up of PEG-His-pLK/KLN25/MM27 and HislPEI/KLN25/MM27, respectively was not totally impeded at DNA/Liposomes/polymer ratio of 1/3/2 (data not shown) while no free pT7-Luc was observed under gel shift assay conditions (Fig. S2.5). mRNA transcription machinery In vitro translation of mRNA-Luc was performed as a function of N/P ratio to evaluate the mRNA accessibility within nanoplexes. The mRNA-Luc used was capped in 3’ and 5’ by the anti-reverse cap analogue ARCA and a Poly(A) tail of 100 adenosine, respectively. For this accessibility test, the luciferase activity produced by mRNA-Luc complexes was measured after incubation with reticulocyte extracts (Fig. 7). The luciferase activity dropped sharply in the case of mRNA-Luc-lPEI and His-lPEI nanoplexes as the N/P ratio increased and it was totally inhibited at N/P ratio of 3. Similar trend in luciferase activity was observed in the case of cationic liposomes (DOSP/MM27 and KLN25/MM27) with complete suppression activity at N/P ratio of 4. As per the gel shift assays results, the observed luciferase activity with mRNA-Luc lipoplexes was ascribed to the amount of free pT7-Luc present in complexes (Fig. S2.6 and S2.7). Thus no translation occurred when mRNA-Luc was strongly complexed with each the vectors. With reference to the transfection of HEK293T7 cells with mRNA-EGFP complexed with lPEI, KLN25/MM27 and LFA, it appeared that efficient transfection was obtained at a N/P ratio that was actually less than the one which totally impeded in vitro transcription of mRNA-Luc (Fig. 4), suggesting the extraction of mRNA from nanoplexes by intracellular biomolecules. Endosome escape Along with the other factors discussed above, the cytoplasmic expression of pDNA and mRNA in HEK293T7 cells also depends on their capability to escape endosomes after


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internalization. Polycations; lPEI, His-lPEI, DOSP/MM27 and KLN25/MM27 exhibit acidmediated membrane destabilization property and consequently, the TE would increase by increasing their amount. Indeed, the number of transfected cells and expression of protein (MFI) with His-lPEI/pT7-EGFP polyplexes increased by 50% and 40% respectively when the N/P ratio increased from 4.25 to 16 (Fig. 8A). Of note, the cytotoxicity did not increase when the N/P ratio increased confirming the low cytotoxicity of His-lPEI. In contrast, the number of transfected cells and MFI decreased with lPEI/pT7-EGFP polyplexes when the N/P ratio increased indicating their cytotoxicity (Fig. 8B). In the case of KLN25/MM27 lipoplexes, the number of transfected cells and MFI increased by 55% and 90%, respectively, when increasing the amount of cationic liposomes (Fig. 8C). This is due to the higher amount of pT7-EGFP complexed with liposomes, (which is in agreement with the gel shift assay) (Fig. S2) and the high concentration of pH-sensitive fusogenic liposomes inside endosomes. Unfortunately, the cytotoxicity increased as well explaining the MFI drop at the N/P ratios > 4. Similar phenomena of MFI drop was observed in the case of DOSP/MM27 lipoplexes with increased liposomes percentage. No cytotoxic effect was however observed (Fig. 8D). The enhanced TE with His-lPEI polyplexes was correlated with an increased uptake of fluorescein-labelled pDNA up to N/P ratio of 3 (Fig. 9A). The post-treatment of those cells with monensin increased fluorescence intensity, revealing that the fraction of fluoresceinlabelled pDNA was inside acidic vesicles. However, in the absence of monensin posttreatment the fluorescence was lower because the fluorescein fluorescence was partially quenched at the acidic pH of endosomes. When a similar experiment was performed with HispLK polyplexes, the uptake of fluorescein-labelled pDNA increased with N/P ratios up to 3 and then drastically dropped due to cytotoxicity (Fig. 9A). The monensin post-treatment induced also an increase of the cell fluorescence intensity, revealing that a part of fluoresceinlabelled pDNA was inside acidic vesicles. When comparing the monensin effect, the



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fluorescence dequenching was lower with His-lPEI polyplexes (< 1.5) than with His-pLK polyplexes (> 2.5) (Fig. 9B). This means that after 4h uptake of His-lPEI polyplexes, pDNA was in a less acidic environment than in the case of His-pLK polyplexes. This low effect of monensin in the presence of His-lPEI is indicative of the presence of a part of pDNA in a neutral compartment such as the cytosol or the nucleus. This is in line with an endosome escape mediated by the protonation of His-lPEI which contains more acid-protonable groups (secondary amine and imidazole groups) than His-pLK. Discussion Stability of nucleic acids, extend of compaction, ease of unpacking and release of nucleic acids are important factors that all together influence the transfection efficiency of nanoplexes. To understand the effect of these factors on the TE of DNA and mRNA, nucleic acids compaction (dye exclusion assay), uptake study, in-vitro transcription and translation, cytotoxicity of nanoplexes and ultimately the TE in HEK293T7 cells were studied. We showed that histidine bearing lipid and polymer based nanoplexes are efficient in cytoplasmic delivery of pDNA and mRNA. Overall, nanoplexes of His-lPEI showed relatively higher TE that signifies possibly the instant unpacking and better membrane destabilization of the endosomes by this polymer. The aim was to evaluate the ease of unpacking and endosomal escape as major cytoplasmic events. Therefore, HEK293T7 cells were used in the study. The transfection of HEK293T7 cells requires the nuclear machinery for the expression of protein which is encoded by pCMV-EGFP. The EGFP expression in this case is significantly different depending on the type of the vector. Though, their endocytosis pathway does not explain these variations. Based on previous studies performed on other cell line (C2C12 cells), we can assume that nanoplexes of LFA, His-lPEI, DOSP/MM27, KLN47 and KLN47/MM27 are internalized in HEK293T7 cells via clathrin-dependent pathway, whereas nanoplexes based on KLN25/MM27 and LPD100 are likely taken up via the caveolae-


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dependent pathway.34 Clathrin-dependent internalization of pDNA nanoplexes of LFA, HislPEI, lPEI or DOSP/MM27 gave significantly higher TE compared to KLN47 and KLN47/MM27. pDNA nanoplexes of KLN25/MM27, LPD100 or LPD16 which are taken up via the caveolae-dependent pathway gave moderate TE. It has been shown that the caveolae join endosomes for cargo delivery.40 High productivity with clathrin-dependent pathway in terms of TE can be explained by the acid-mediated membrane destabilization feature of the components, which favours the pDNA cytoplasmic delivery. On the other hand, in the caveolae-dependent uptake, a lower amount of complexes that join endosomes is possibly responsible for the moderate TE. The TE differences can be attributed to the nuclear import of pDNA, although, the transfection here is in actively dividing cells thus the variations are rather due to cytoplasmic events including endosome escape and/or dissociation of pDNA nanoplexes. The transfection of HEK293T7 cells with pT7-EGFP nanoplexes under the same conditions does not globally modify the TE pattern. HEK293T7 cells allow gene expression of pDNA encoding gene in the cytosol under the T7 promoter due to the T7 RNA polymerase machinery which is enormously present in the cytoplasm and absent in the nucleus of this particular cell line.

41, 42

In this study, it was found that the EGFP expression level (as

determined from MFI) with pT7-EGFP is 10-fold lower than with pCMV-EGFP. The T7 RNA polymerase machinery produces uncapped mRNA-EGFP having no PolyA tail. The mRNA of such kind has short half-life and produces low level of EGFP. This reasoning explains very well the low expression with pT7-EGFP. Considering the case of mRNA transfection of HEK293T7 cells with mRNA-EGFP nanoplexes, the results are well consistent and quite similar to those of the transfection with pDNA (pT7-EGFP) nanoplexes. Interestingly, though the pT7-EGFP produces mRNA-EGFP without a cap and a polyA tail, its EGFP expression after transfection was always higher than the transfection with the



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synthetic mRNA-EGFP bearing a cap and a long polyA tail (that expected to be more stable and better translated). It means that the number of mRNA-EGFP transcripts produced after DNA transfection is higher than the available one after internalization of the synthetic mRNA-EGFP. Globally, the variation in TE of pCMV-EGFP, pT7-EGFP or mRNA-EGFP with different vectors shows the variation in the extent of endosome escape and/or the dissociation rate of the complexes. The expression of pDNA and synthetic mRNA depends on the capacity of their vector to mediate the escape from endosomes after internalization. This process involves the protonation of the secondary amines and imidazole groups that eventually induces membrane destabilization which is usually attributed to the “proton sponge” effect. 43 Based on the wellknown chloroquine effect, it was supposed that the “proton sponge” effect induces the neutralization of the protons generated in the lumen of endosomes or lysosomes by the VATPase pump, therefore increasing the entrance of chloride ions and water. This leads to osmotic swelling and rupture of the vesicle membrane allowing pDNA delivery in the cytosol. In fact, “proton sponge” effect has never been demonstrated in cellulo with polyplexes and lipoplexes. Though pH changes have been reported in vesicles containing fluorescent PEI and imidazole-cyclodextrin supporting the neutralizing effect related to polymer protonation, yet it was not possible to correlate the transfection efficiency with the high buffering capacity of the vector.44 Benjamissen et al. attempted to measure pH changes in acidic vesicles by using pH nanosensors upon PEI internalizations and come to the conclusion that no change in the pH was detectable in HeLa cells.45 Thus, the “proton sponge” effect remains questionable. Though endosomal escape is considered a critical step for transfection, other intracellular events including pDNA transport in the cytosol and its nuclear import are equally important. Another possibility of endosomal escape would be that protonated PEI interacts with anionic lipids which are present in the cytoplasmic-facing side of endosomal membrane and


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destabilizes the membrane through pores formation and/or surfactant like effect. Several evidences from electron microscopy images,46 real-time visualization study47 and cell patchclamp experiments48 are now supporting this hypothesis. Since polyhistidine has been shown to promote membrane fusion49 and histidine-rich peptides showed membrane permeation50 in acidic medium, both these mechanisms are likely involved in the endosomal escape of HislPEI polyplexes. Theoretical investigations predict that a certain quantity of free PEI inside endosomes facilitates membrane rupture after the acidification of the vesicle lumen.51 Free PEI and His-lPEI in endosomes may come from polyplexes dissociation and/or an excess of polymer present in the polyplexes solution. The hypothesis of PEI polyplexes dissociation in endosomes was supported by a real-time visualization study that confirmed that PEI and pDNA are delivered separately from endosomes into the cytosol.47 Moreover, it has been reported that a large amount of PEI dissociates from pDNA in early stage of transfection process52 and could intercalate in the membrane to form the pores.48 In the case of His-lPEI, we observed an increased TE of pDNA in the presence of an excess of His-lPEI. This TE enhancement confirms the membrane-destabilising ability of the free polymer. This would also mean that His-lPEI polyplex stability could be a limiting factor for a better endosome escape. In the case of lPEI polyplexes, it was not possible to obtain the same effect because of lPEI cytotoxicity. No significant improvement in TE was observed by increasing the amount of cationic liposomes in lipoplexes, indicating that the amount of liposomes within endosomes was sufficient to induce endosomal escape in contrast to polyplexes. In the case of lipoplexes, it is strongly suggested that the destabilization of the endosomal membrane occurs via lipid mixing or membrane fusion between liposomes and the membrane leading to the delivery of nucleic acids in the cytosol. Once internalized into the endosomes, positively charged lipoplexes electrostatically interact with the anionic lipids of endosomal membrane. In this



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process, the anionic lipids laterally diffuse into the lipoplexes and form neutral ion pairs with the cationic lipids and thus remove the nucleic acid from the complex.47 The free nucleic acids come out into the cytosol from the fused structure. In KLN25/MM27 and DOSP/MM27 liposomes, MM27 bearing a histamine polar head is an acid-fusiogenic lipid as previously shown.30 The fusion of lipoplexes with the endosome membrane can be enhanced with those liposomes which contain histidylated co-lipids such as MM27. As discussed above, the dissociation of nucleic acids from the vectors seems to occur before or during endosome escape.47,

48, 52

Therefore, the accessibility of pT7-DNA and

mRNA to the transcription and translation machinery should not be impaired in cellulo. Indeed, in vitro transcriptional activity of pT7-Luc is dramatically reduced when pDNA is condensed with the vectors. This is in line with previous study describing that the transcription and translation of pDNA in lipoplexes are greatly impaired compared to naked DNA and further transcriptional activity of pDNA was completely inhibited at N/P ratios > 3:1 in lipoplexes of DOTAP.53 Furthermore, the transcriptional activity was hardly detected at N/P ratio of 2.2 when in vitro transcription of bPEI/pDNA-Luc complexes was performed with basal transcriptional machinery.46 However, transcriptional activity occurred with bPEI/pDNA and lPEI/pDNA complexes when cytosolic extract was used, suggesting that pDNA can be extracted from PEI polyplexes by intracellular biomolecules. Here, the transcription machinery of pT7-DNA in vitro is dramatically less impeded in His-lPEI/pDNA complexes. This could be correlated with the cryo-TEM observations that reveal a spherical shape and amorphous character of His-lPEI polyplexes, suggesting that the binding energy between DNA and His-lPEI would be weaker compared to the binding energy in lPEI polyplexes.54 Likewise, the in vitro translation activity of mRNA-Luc was completely suppressed regardless of the type of complex used including His-lPEI. Conclusion


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The cytoplasmic transcription of pDNA with HEK293T7 cells gives us a common cytoplasmic region for the expression of both mRNA- and pDNA nanoplexes. We effectively found out that PEI, histidine bearing lipid and histidine-rich polymer based nanoplexes are efficient in pDNA and mRNA unpacking, the endosomal escape and their accessibility to the transcription machinery in the cytoplasm, presumably thanks to the influence of their acidprotonable groups. Globally, mRNA transfection profile is well corroborative with the cytoplasmic transfection of pT7-pDNA as well as with the nuclear transfection with pCMVDNA. Overall, nanoplexes consisting of His-lPEI showed relatively higher TE thanks to the free polymer-mediated endosome destabilization. The unpacking of the complex inside endosomes would intensify this destabilization process, allowing high availability of pDNA for its transport towards the nucleus.

Acknowledgements This work was supported by grants from Association Française contre les Myopathies (Projet Stratégique 2009, n°15628, AFM, Evry, France). We thank the P@CYFIC platform at CBM Orléans. We certify that there is no conflict of interest, no competing of interest and no disclosure.

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Figure captions Figure 1: Diagram showing the intracellular ways leading to protein expression from plasmid DNA or mRNA in HEK293T7 cells transfected either with pCMV-DNA, pT7-DNA or mRNA complexes. Figure 2: TE of various pDNA complexes. HEK293T7 cells were transfected with 2.5 µg (A) pCMV-EGFP or (B) pT7-EGFP complexed with the indicated vectors. The figure shows the percentages of EGFP positive cells (black bars), the mean fluorescence intensities (MFI) (white bars) and the cytotoxicities (dashed bars. MFI are expressed relative to the MFI values of His-lPEI complexes, considered as 100 %. N/P is the charge ratio where N is the number of positive charges of polymer or lipid and P is the number of phosphate charges of pDNA. The N/P ratios of pDNA complexes with PEG-His-pLK, His-lPEI, IPEI, KLN47, KLN47/MM27, KLN25/MM27, DOSP/MM27, LFA, LDP100 and LPD16 were 2.5, 4, 4, 4, 1, 4, 6, 13, 4.3 and 4.2, respectively. Values are means ± SD of three different experiments. Figure 3: Comparative global TEs of the nuclear (pCMV-EGFP) (white bars) and cytoplasmic (pT7-EGFP) (black bars) gene expression systems. The global TE is presented as the number of EGFP positives cells x MFI. Values are means ± SD of three different experiments. Figure 4 : TE of mRNA complexes. HEK293T7 cells were transfected with 5 µg mRNAEGFP complexed with the indicated vectors. The figure shows (A) the percentages of EGFP positive cells, (B) the mean fluorescence intensities (MFI) and (C) the global TE i.e. the number of EGFP positives cells x MFI. MFI are normalized to the MFI value of His-lPEI complex, considered as 1000 A.U. The N/P ratios of pDNA complexes with PEG-His-pLK, His-lPEI, PEI, KLN47, KLN25/MM27, DOSP/MM27, LFA, LDP100 and LPD16 were 2.5,



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5, 1.5, 4, 1.8, 4, 3.3, 4.3 and 4.2, respectively. Values are means ± SD of three different experiments. Figure 5: Comparative TE of pT7-EGFP (black bars) and mRNA-EGFP (white bars) complexed with the indicated vectors. The figure shows (A) the percentages of EGFP positive cells, (B) the mean fluorescence intensities (MFI), normalized to the MFI value of His-lPEIpT7-EGFP polyplex, considered as 1000 A.U. and (C) the global TE i.e. the number of EGFP positives cells x MFI. Values are means ± SD of three different experiments. Figure 6: Dye exclusion assay. The fluorescence intensity extinction of 1 µg/mL pDNA in the presence of EtBr was measured as a function of (●) His-lPEI, (▲) lPEI, (∆) pLK, (□) DOSP/MM27 or (■) KLN25/MM27 amount. N/P is the charge ratio. Values are means ± SD of three different experiments. Figure 7: In vitro transcription/translation of pT7-Luc complexes and mRNA-Luc. pT7Luc (∆) or mRNA-Luc (●) (0.2 µg) free or complexed with the indicated vectors at various N/P charge ratio was mixed with the TNT Quick Coupled Transcription/Translation Systems Kit (Promega). After 1 h 30 min incubation at 30°C, the luciferase activity was measured and expressed as the percentage of control which refers to the values of luciferase activities of pT7-Luc plasmid complexes compared to the value of free pT7-Luc plasmid. The transfection values shown are average of three independent experiments. (▲) Percentage of pDNA complexes determined from dye exclusion assays (Fig. 6). Figure 8: Influence of the N/P charge ratio on the TE of pT7-EGFP/His-lPEI polyplexes. HEK293T7 cells were transfected with 2.5 µg pT7-EGFP complexed either with (A) HislPEI, (B) lPEI, (C) KLN25/MM27 or (D) DOSP/MM27 at various N/P charge ratios. The figure shows (■) the percentages of EGFP positive cells, (□) the mean fluorescence intensities (MFI), normalized to the MFI value of His-lPEI complex at N/P ratio of 16, considered as 100 30 ACS Paragon Plus Environment

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% and (▲) the cytotoxicity, normalized to untreated cells. Values are means ± SD of three different experiments. Figure 9: Influence of the N/P charge ratio on the uptake of polyplexes. HEK293T7 cells were incubated for 4 h with 2.5 µg fluorescein-labelled pCMV-Luc complexed with His-lPEI (●, ○) or His-pLK (▲, ∆) at various N/P charge ratios. The mean fluorescence intensities (MFI) of the cells were measured by flow cytometry before and after a post-treatment with monensin. (A) The cell fluorescence intensities are normalized to the highest value. (B) Effect of monensin treatment for His-lPEI (●) and His-pLK (▲). MO+ and MO- are the fluorescence intensities of the cells after and before the monensin treatment, respectively. Values are means ± SD of three different experiments. Supplementary data Figure S1: Structures of lipids and polymers used: (a) histidinylated lPEI (His-lPEI); (b) PEGylated

and

histidylated

methylimidazolium

polylysine

iodide)propylene)

(PEG-His-pLK);

phosphoramidate

(c)

O,O-dioleyl-N-(3N-(N-

(KLN25);

(d)

O,O-dioleyl-

phosphoramidate arsonium (KLN47); (e) dioleyl succinyl paromomycin (DOSP); (f) O,Odioleyl-N-histamine phosphoramidate (MM27). Figure S2.1 to S2.6: Agarose gel shift assay of pDNA and mRNA as complexes with different vectors at various N/P. N/P= 0; indicate the free pDNA or mRNA.



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Table 1: Size, polydispersity index (PDI), ζ-potential of DNA and mRNA nanoplexes DNA-Nanoplexes

2.5 µg pDNA

Particle size (nm±SD)

PDI (mean±SD)

ζ-potential (mV±SD)

Polyplexes N/P PEG-His-pLK 2.5 88.2±0.7 0.3±0.05 +14.7±0.7 His-lPEI 1/6 4 103.7±0.6 0.3±0.07 +17.0±0.3 IPEI 1/2 4 90.2±0.9 0.1±0.06 +21.3±0.6 Lipoplexes KLN47 4 183.4±0.6 0.3±0.2 +09.4±0.5 KNL47/MM27 1 178.1±1.2 0.2±0.009 +17.3±0.8 KLN25/MM27 4 186.9±1.8 0.3±0.2 +12.3±0.2 DOSP/MM27 1/4 6 203.1±0.7 0.4±0.5 +15.4±0.6 LFA* 13 140±0.7 0.3±0.1 +19.7±0.3 Lipopolyplexes LPD100 4.3 172.2±0.5 0.2±0.06 +13.7±0.3 LPD16 4.2 167.8±1.3 0.2±0.1 +18.3±0.6 mRNA-Nanoplexes 5 µg mRNA Polyplexes N/P PEG-His-pLK 2.5 90.7±1.5 0.2±0.1 +11.9±0.5 His-lPEI 5 122.9±0.9 0.3±0.05 +13.5±0.8 IPEI 1.5 94.7±1.2 0.2±0.2 +19.4±0.2 Lipoplexes KLN47 4 173.2±1.7 0.3±0.1 +11.3±0.6 KNL47/MM27 1 159.5±0.6 0.2±0.06 +15.2±0.5 KLN25/MM27 4 163.2±1.8 0.3±0.1 +11.4±0.5 DOSP/MM27 6 178.6±0.8 0.2±0.7 +13.8±0.9 LFA* 13 135.9±1.7 0.2±0.3 +18.9±0.7 Lipopolyplexes LPD100 4.3 169.2±0.9 0.2±0.04 +15.2±0.8 LPD16 4.2 175.9±0.8 0.2±0.09 +16.8±0.6 *LFA: Lipofectamine2000™ used as the positive transfection control with DNA and mRNA.


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254x190mm (96 x 96 DPI)

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Intracellular Availability of pDNA and mRNA after Transfection: A

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