Compaction and Transmembrane Delivery of pDNA: Differences

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Compaction and transmembrane delivery of pDNA: differences between l-PEI and two types of amphiphilic block copolymers Alexander Raup, Hui Wang, Christopher V. Synatschke, Valerie Jerome, Seema Agarwal, Dmitry V. Pergushov, Axel H.E. Mueller, and Ruth Freitag Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01678 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017

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Compaction and transmembrane delivery of pDNA: differences between l-PEI and two types of amphiphilic block copolymers

Alexander Raupa, Hui Wangb, Christopher V. Synatschkeb,d , Valérie Jérômea, Seema Agarwalb, Dmitry V. Pergushovc, Axel H.E. Müllerb,e, Ruth Freitaga,* a

Process Biotechnology and bMacromolecular Chemistry II, University of Bayreuth, 95440

Bayreuth, Germany c

Department of Polymer Science, School of Chemistry, M.V. Lomonosov Moscow State

University, 119991 Moscow, Russia d

present address: Simpson Querrey Institute for BioNanotechnology, Northwestern University,

Chicago, IL 60611, USA e

present address: Institute of Organic Chemistry, Johannes-Gutenberg-University, 55099 Mainz,

Germany *corresponding author, email address: [email protected], postal address: Process Biotechnology, University of Bayreuth, 95440 Bayreuth, Germany

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ACS Paragon Plus Environment

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Abstract Polycations are popular agents for non-viral delivery of DNA to mammalian cells. Adding hydrophobic, biodegradable, or cell penetrating functions could help to improve their performance, which at present is below that of viral agents. A crucial first step in gene delivery is the complexation of the DNA. The characteristics of these ‘polyplexes’ presumably influence or even determine the subsequent steps of membrane passage, intracellular travelling / DNA release, and nuclear up-take. Herein, polyplexes formed with linear poly(ethylenimine) (l-PEI) are compared to complexes generated with functionalized diblock copolymers. While l-PEI interacts only electrostatically with the DNA, interaction in case of the diblock polymers may be mixedmode. In certain cases transfection efficiency improved when the polyplexes were formed in hypertonic solution. Moreover, whereas conventional PEI-based polyplexes enter the cells via endocytosis, at least one of the diblock agents seemed to promote entry via transient destabilization of the plasma membrane.

Keywords gene delivery, cellular uptake, PDMAEMA, l-PEI, PHMG-PCL block copolymers

1. Introduction Gene delivery to mammalian cells (‘transfection’) requires the uptake of plasmid DNA (pDNA) by the cells followed by transfer into the nucleus. Since cells do not take up naked pDNA, a delivery agent is required. More than 25 years ago, Boussif et al. first proposed the polycation poly(ethylenimine) (PEI) for gene delivery, which to date is still the ‘gold standard’ in the area1. Some other polycations have been discussed, most prominently, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)2. The first task of the polycationic gene delivery agent is to

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condense the negatively charged pDNA into a positively charged “polyplex” small enough for cellular uptake, most likely by endocytosis3. In this context, the superior performance of PEI as transfection agent is explained by a presumed ability to escape the degrading environment of the endosome via the ‘proton sponge effect’4, 5. Over the years some basic design principles have been derived correlating the structure of a polycationic transfection agent to its performance. In general, both the transfection efficiency and the cytotoxicity increase with the size6, 7. In consequence, an optimal size exists, for which a large fraction of the cells become transfected, while most still survive the procedure. For a given size, non-linear polymers were shown to be more efﬁcient and less cytotoxic than the corresponding linear ones8-10. Based on this principle, our group has recently introduced star-shaped polycationic nano-structures, which not only performed well in standard experiments, but showed some ability to transfect cells, which before then had been not considered susceptible to non-viral transfection, examples include differentiated and/or non-dividing cells11. To date no explanation for this ability has been put forward. Some additional features have been proposed by various authors to improve or modify the performance of polycationic delivery agents. While electrostatic interaction is the basis for polyplex formation, DNA is also capable of hydrophobic interaction. By adding hydrophobic domains to the delivery agents, a mixed-mode type of interaction can be produced, which can be fined-tuned to have a beneficial effect on polyplex formation and stability12-15. Secondly it has been put forward that the biocompatibility of the delivery agent can be improved by making it biodegradable, probably one of the most interesting attempts to reduce the cytotoxicity of polycationic transfection agents in recent years16-20. In this context, the introduction of a polycaprolactone (PCL) block is a very elegant suggestion, as this block would be both hydrophobic and biodegradable. A considerable reduction of the cytotoxicity has been 3


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demonstrated for block copolymers of PEI and PCL16 and ascribed to the biodegradability of the PCL block by the authors. Finally, a biomimetic approach has been suggested to change the mechanism of cellular entry. Cell penetrating peptides (CPPs) are one of nature’s solutions for the transmembrane delivery of large cargo molecules20. In these structures, positively charged guanidine groups are crucial to the penetration ability20, 21. Such groups could also be introduced into synthetic polycations. While interesting, none of these ideas has to date led to an improved commercially available transfection agent. Admittedly, a univocal demonstration of their usefulness is difficult, since a series of processes is affected, reaching from complex formation to transgene delivery. This contribution takes a detailed look on the influence of such structures at the level of DNAcomplexation and transmembrane delivery. Transfection efficiencies and cytotoxicities are then discussed in view of these results. Two types of complex delivery agent were investigated. One was a poly(1,2-butadiene)-block-PDMAEMA diblock copolymer (m-PDMAEMA)11, which consists of a hydrophobic poly(butadiene) block and a charged PDMAEMA block. As previously shown22, m-PDMAEMA forms stable star-shaped micelles in aqueous solution. The strongly hydrophobic poly(butadiene) blocks presumably form the core of these micelles and should not be available for interaction with dissolved molecules including DNA. The second diblock copolymer was a poly(hexamethylene guanidine hydrochloride)-block-poly(ε-caprolactone), PHMG-b-PCLx23, a structure, which combined a charged poly(hexamethylene guanidine hydrochloride) block (electrostatic interaction, but also cell penetration) with a hydrophobic poly(ε-caprolactone) block (hydrophobic interaction, but also biodegradability). All experiments were referenced to l-PEI (25 kDa), an established polycationic transfection agent. Two cell lines were studied, one human (HEK-293), one rodent (CHO-K1) to get a first inclination of putative cell specific effects. 4


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2. Materials and methods 2.1. Materials. Cell culture materials were from Greiner bio-one (Frickenhausen, Germany), chemicals from Sigma-Aldrich (Taufkirchen, Germany). Fetal calf serum (FCS) was from Biochrom AG (Berlin, Germany), Dulbecco’s Phosphate-Buffered Saline without Ca2+ and Mg2+ (DPBS) from Lonza (Visp, Switzerland). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered glucose (HBG, 20 mM HEPES, 5 wt% glucose, pH 5.5) was prepared in house and sterilized by filtration (0.2 µm, cellulose acetate filters). Cell culture media “R10” (Roswell Park Memorial Institute (RPMI) medium 1640 without glutamine, supplemented with 10 vol% FCS, 2 mM Lglutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin), Medium Eagle (MEM) media MEM10 (MEM Earle’s without L-glutamine / FCS, supplemented with 10 vol% FCS, 4 mM Lglutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin) and Opti-MEM (NaCl: 116 mM) were from Lonza (Visp, Switzerland), Biochrom AG (Berlin, Germany), and Thermo Fisher Scientific (Dreieich, Germany). For the cytoxicity test (“MTT test”) MEM Earle’s medium (Thermo Fisher Scientific, Dreieich, Germany) without L-glutamine and without phenol red was supplemented with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 1 mg/mL) and sterilized by filtration. 2.2 Plasmid pEGFP-N1 (4.7 kb, Clontech Laboratories, Inc., Mountain View, CA, USA) encoding for the enhanced green fluorescent protein (EGFP) driven by the cytomegalovirus immediate early promoter, was amplified in E. coli DH5α (lysogeny broth (LB) medium) using standard laboratory techniques. The EndoFree Plasmid Kit (Giga Prep) from QIAGEN (Hilden, Germany) was used for purification (quality control: > 80% supercoiled topology (agarose gel) and 5


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A260/A280 ≥ 1.8). Purified plasmids were solubilized in sterile cell culture-grade water (SigmaAldrich, Germany) and stored at -20°C. 2.3 Cells CHO-K1 (CCL-61, American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel, Germany) and HEK-293 (CRL-1573, ATCC) cells were maintained in R10, the L929 cells (murine fibroblast, CCL-1, ATCC) used in the MTT assay in MEM10. Cells were cultivated at 37°C in a humidified 5% CO2 atmosphere. 2.4 Transfection agents Linear poly(ethyleneimine) (l-PEI, 25 kDa) was from Polysciences Inc. (Warrington, PA, USA). The two amphiphilic polymers, poly(1,2-butadiene)-block-PDMAEMA (m-PDMAEMA) and poly(hexamethylene guanidine hydrochloride)-block-poly(ε-caprolactone) (PHMG-b-PCLx) were prepared in house as previously published23, 24. Full details on synthesis protocols and structural characterization can be found in the cited publications. m-PDMAEMA (Mn 54 kDa, polydispersity < 1.07) contains an average of 290 monomeric units in the butadiene block and 240 in the PDMAEMA block. In aqueous solution m-PDMAEMA forms stable micelles (hydrodynamic radius, Rh: 27 nm, number of aggregation, Nagg: 120). The three investigated PHMG-b-PCLx copolymers contained a 9-unit PHMG block together with a PCL block of varied length (x = 5, 6, 12), Table 1. Table 1. Poly(hexamethylene guanidine) hydrochloride-block-poly(ε-caprolactone) Polymer

number of units in the PCL block

Time of polymerization

Mn

PDI

a

PHMG-b-PCL12

12

24 h

2700 Da

1.3

a

PHMG-b-PCL6

6

8h

2000 Da

1.2

5

4h

1900 Da

1.2

PHMG-b-PCL5 a)

23

From Wang et al 2014 , PDI Polydispersity index (Mw/Mn) All structures contain nine hexamethylene guanidine units in the PHMG block. Characterization in regard to size and distribution of the blocks, as previously published23. 6


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2.5 Transfection protocol Transfection was done according to the previously published standard protocols for adherent cells25. Briefly, CHO-K1 cells were seeded into 6-well culture plates at 2 × 105 cells per well in 2 mL growth medium (R10) 24 h prior to transfection. One hour before transfection, cells were rinsed with DPBS, supplemented with 1 mL Opti-MEM, and put back into the incubator. Polyplexes were prepared in a final volume of 200 µL by first diluting the desired amount of pDNA stock solution (1.2 – 4.3 µg/Well) with the indicated diluent (NaCl solution or HBG buffer) followed by the addition of sufficient amounts of the concentrated polymer solution to achieve the intended N/P-ratio (polymer N to DNA P). The mixture was vortexed for 10 s and incubated for 20 min at room temperature. 1 mL Opti-MEM was added, followed by vortexing and 10 min incubation at room temperature. The polyplex mixture (1.2 mL total volume) was added drop-wise to the cells and distributed by gently rocking the plate. After an incubation period of 4 h the supernatant was removed from the cells and replaced by 2 mL of fresh growth medium. HEK-293 cells, which adhere only weakly and where washing would have led to considerable losses, were harvested and reseeded as described above, but then maintained under regular growth conditions right up to transfection. The supernatant was cautiously aspirated and immediately replaced by 1.2 mL of the polyplex mixture in Opti-MEM. Subsequently, HEK-293 cells were treated as the CHO-K1 cells. 2.6. Physico-chemical characterization of the polyplexes Polyplexes for the physico-chemical characterization were formed as for transfection. Their size was determined by non-invasive back scattering (Zetasizer: Nano ZS, Malvern, Herrenberg, Germany) utilizing a He−Ne laser (λ = 633 nm, max = 5 mW). Hydrodynamic radii were followed for 20 min. Then polyplexes were diluted 6-fold with Opti-MEM and further monitored. 7


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All experiments were conducted at 25°C. Zeta-potentials of the polyplexes were determined using the same instrument. The gel retardation assay was done in 1% (w/v) agarose gels with Tris-acetate-EDTA as running buffer (running time 90 min, applied voltage 90 V). Loading buffers with and without bromophenol blue were used. Gels were stained with ethidium bromide (EtBr, 10 µg/mL) and the DNA visualized under UV light (254 nm). The melting temperatures of the complexed vs. free pDNA were measured as previously described26, using a standard real time PCR machine (MX3005P, Agilent Technologies, Heidelberg, Germany) equipped with an SYBR green filter (BP 504 (12 nm)). Samples containing 25 µg/mL pDNA were mixed with 5 µl of a 5X SYBR green I solution prepared in NaCl or HBG (total volume: 50 µL) and incubated for 10 min. The polymers were added and the solution vortexed for 10 s followed by 30 min incubation at room temperature in the dark. The melting temperature of the DNA was defined as the inflection point of the sigmoidal fluorescence-versus-temperature curve (fluorescence normalized to the starting one at 25 °C). 2.7 Analysis of the transfection results Cytotoxicities were tested by standardized test (MTT text according to ISO 10993-527) using L929 murine fibroblasts. Transfection efficiencies (percentage of cells expressing the fluorescent transgene EGFP) were quantified by flow cytometry (Cytomics FC500, Beckman Coulter, Krefeld, Germany, 488 nm). Cells were harvested by trypsinization, recovered by centrifugation (200 g, 5 min) and resuspended in 500 µL DPBS containing 1 µg/mL propidium iodide (PI) to counterstain the dead cells. Cells were initially evaluated by scatter properties (forward scatter / side scatter (FSC/SSC)) to select a region representing single, non-apoptotic cells. Transgene expressing cells were classified as low producers: fluorescence intensity between 1 and 10 a.u.; middle producers: 8


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fluorescence intensity between 10 and 100 a.u. and high producers: fluorescence intensity > 100 a.u. in the non-apoptotic, gated cell population. Membrane permeabilization was demonstrated via the cellular uptake of the normally not membrane penetrable DNA intercalating dye PI (1 µg/mL) during the course of the transfection. Cells incubated with PI in the absence of polymers/polyplexes served as negative controls. Positive controls were cells treated with Digitonin (5 µg/mL, 5 min), a well-known reversible cell permeabilization reagent28. PI uptake by the cells was quantified by flow cytometry. PI+-cells were classified as PIlow: fluorescence intensity between 0.8 and 20 a.u. and PIhigh: fluorescence intensity > 20 a.u. cells. PIhigh cell were considered dead cells. Intercalation of PI into polyplexed DNA was analyzed using a plate reader plate (fluorescence mode (Ex: 535 nm/ Em: 612 nm), GENios Pro, Tecan Deutschland GmbH, Crailsheim, Germany). The relative fluorescence was calculated as follows: Relative Fluorescence =

( − ) ( − )

With Fobs: sample fluorescence, F0: fluorescence of the same concentration PI not intercalated into DNA, FDNA: fluorescence of the same concentration PI fully intercalated into free DNA. 2.8 Statistical analysis Group data are reported as mean ± SD. Statistical evaluation of the MTT results was with SigmaPlot 11.0 (Systat Software GmbH). The one-way ANOVA with Bonferroni t-test was used to determine whether data groups differed significantly from each other. Statistical significance was defined as p < 0.05.

3. Results and discussion

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Three types of polymeric transfection agent were investigated, a linear polycation (l-PEI, 25 kDa), which is a commercially available standard vector for the transfection and mammalian cells, and two more complex structures. One was a poly(1,2-butadiene)-block-PDMAEMA diblock copolymer (m-PDMAEMA)11, which consists of a hydrophobic poly(butadiene) block and a charged PDMAEMA block (Mn 54 kDa). In aqueous solution, m-PDMAEMA forms large, stable micelles (hydrodynamic radius 27 nm) with the hydrophobic poly(butadiene) blocks presumably forming the core of these micelles. The second investigated complex structure was poly(hexamethylene guanidine hydrochloride)-block-poly(ε-caprolactone), PHMG-b-PCLx23, which combines a charged poly(hexamethylene guanidine hydrochloride) block (electrostatic interaction, but also cell penetration) with a hydrophobic poly(ε-caprolactone) block (hydrophobic interaction, but also biodegradability). In case of PHMG-b-PCLx no experimental evidence could be found for micelle formation in aqueous solution.

3.1. Colloidal properties of the polyplexes Charge is an important factor for cellular uptake of particles via endocytosis mediated by proteoglycans such as heparin sulfate1, 29.

Figure 1. Zeta potentials of the polyplexes as a function of the N/P-ratio

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m-PDMAEMA (), PHMG-b-PCL5 (), l-PEI (), an unbuffered aqueous NaCl solution (150 mM) was used as matrix, lines serve as guides to the eye.

Figure 1 summarizes the zeta potentials of polyplexes formed between the pDNA (15 µg) and increasing amounts of m-PDMAEMA, PHMG-b-PCL5, or l-PEI. According to these data, in the case of m-PDMAEMA an N/P-ratio of at least 3 is required to charge-compensate the DNA, while the N/P-ratio should be at least 7.5 in case of PHMG-b-PCL5. For l-PEI an N/P-ratio of 4.5 is necessary for charge neutralization according to these experiments. When the N/P-ratio is increased further, the zeta potential typically reaches a plateau. Plateau values of approximately 10 mV were observed here for the two diblock polymers, whereas the values for l-PEI were close to 20 mV. In case of m-PDMAEMA the plateau was reached for N/P > 5, in case of PHMG-bPCL5 for N/P > 10, and in case of l-PEI for N/P > 7. The fact that in case of PHMG-b-PCL5 the highest N/P-ratios are required for charge compensation and reaching the charge plateau argues that among the three investigated polycations, PHMG-b-PCL5 forms the polyplexes with the lowest binding strength, which is most likely due to the small size of this polycation. While a positive net charge is required for interaction and cellular uptake, there is no evidence that a high positive surface net charge in general is beneficial. Quite the opposite, in view of a potential in vivo application, polyplexes with a lower positive surface charge may have advantages as they show fewer tendencies to trigger erythrocyte agglomeration30. In this regard polyplexes formed by the two diblock copolymers (plateau zeta potentials 10 mV) may have advantages compared to l-PEI (20 mV). Figure 2 summarizes the development of the polyplex size with time (monitored by dynamic light scattering). Previously it had been shown that polyplex size does not depend on the N/Pratio, provided the latter is from the plateau region of the zeta potential curve. The N/P-ratios 11


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chosen for m-PDMAEMA (7.5) and l-PEI (20) were thus taken from the standard transfection protocol, the value for PHMG-b-PCL5 (34) was determined in a preliminary test (data not shown). To investigate effects of ionic strength, polyplexes were prepared in unbuffered aqueous NaCl matrices of varied concentration (150, 300, 600, 900 mM), but also in HEPES-buffered glucose (HBG, 20 mM HEPES, 5 wt% glucose, pH 5.5). Note that an unbuffered 150 mM NaCl solution is the standard matrix called for in our transfection protocol. After 20 min all mixtures were diluted 6-fold with Opti-MEM to mimic transfection conditions. Our results fit well with previous observations showing that the matrix selected for polyplex formation has a marked effect on polyplex size31. More interestingly however, significant differences are seen between the investigated polymers. In case of l-PEI (Figure 2A) and PHMGb-PCL5 (Figure 2B) the polyplexes with the largest hydrodynamic radii were obtained in 150 mM NaCl, while this value decreased at higher NaCl concentration. This dependency of the polyplex size on the NaCl concentration can be considered typical for standard polycationic transfection agents and has been described before for l-PEI31.

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Figure 2. Dynamic development of the polyplex size (hydrodynamic radius, Rh) during incubation A) l-PEI, N/P 20, B) PHMG-b-PCL5, N/P 34, C) m-PDMAEMA, N/P 7.5, The polyplexes were prepared in HBG () or unbuffered aqueous NaCl solutions (150 mM , 300 mM , 600 mM , 900 mM ) as matrix. The initial volume was 200 µL, after 20 min 1 mL of Opti-MEM was added (symbol color switched to grey to facilitate distinction). 13


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In all investigated NaCl matrices polyplex an increase in size, indicative of polyplex aggregation, was observed during the initial 20 min of incubation, whereas once the mixture was diluted 6fold with medium, the average polyplex size started to decrease again approaching identical final values of around 200 nm in case of l-PEI and of approximately 130 nm in case of PHMG-bPCL5. In view of an eventual application for cellular transfection, this is beneficial, as for the delivery of pDNA into cells and tissues small polyplexes (< 200 nm) have been shown to be much more effective32-34. However, shrinking takes time (at least another 20 min in case of l-PEI, more rapidly in case of the smaller PHMG-b-PCL5), during which the complex size changes continuously. This dynamic situation may contribute to the fact that keeping exact incubation/maturation times is known to be crucial for success in l-PEI transfections. Moreover, in view of these data, the lengthy initial “maturation period” suggested in most transfection protocols involving PEI as transfection agent and 150 mM NaCl as matrix seems counterproductive. Extremely small polyplexes (33 nm for l-PEI, 40 nm for PHMG-b-PCL5) were produced in HBG as matrix. Moreover, polyplexes were stable and did not aggregate in that matrix. After dilution with medium, the size of these polyplexes also started to approach the end values of 200 nm and 130 nm for l-PEI and PHMG-b-PCL5 respectively. It is possible that the better results reported for HBG rather than NaCl as matrix for polyplex formation in l-PEI transfections31 is due to this difference in complex size development. A distinctly different behavior was seen for the polyplexes formed with m-PDMAEMA, Figure 2C. Regardless of the matrix and the subsequent dilution, these complexes had a constant and stable hydrodynamic radius of approximately 100 nm. HBG is a matrix of low ionic strength. This means little charge shielding and therefore strong attractive interactions between the oppositely charged macromolecules within the polyplexes and 14


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strong electrostatic repulsion between the individual charged polyplexes. This would explain why polyplexes formed in this matrix are small and stable. The predominant effect of NaCl is charge shielding leading to a weakening of the electrostatic interactions. Concomitantly, the strength of hydrophobic interactions should increase with increasing salt concentration. However, this seems to be of little consequence here, where the simple polycation PEI and PHMG-b-PCL5, a polymer with a presumably exposed hydrophobic block, show essentially the same behavior. m-PDMAEMA also contains a hydrophobic block. However, according to our current understanding, these blocks form the micellar core and are not available for interaction with the DNA. Instead we propose that the large micellar structures (Mn = 54 kDa, aggregation number 120 corresponding to a micelle size of 6.5 mio Da) are much less likely to participate in the bridging and cross-linking events leading to polyplex aggregation in case of l-PEI and PHMG-bPCL5. Polyplex aggregation or lack thereof would also explain, why polyplex sizes decrease again once the mixture has been diluted with Opti-MEM. The exact composition of the medium is proprietary, however, any cell culture medium contains surface-active components that counteract aggregation. The end value for polyplex size correlates well with the NaCl concentration of Opti-MEM, which according to the supplier is 116 mM.

3.2. Gel retardation assay The gel retardation assay is a tool to determine the N/P-ratio required for complete DNA complexation. As long as free negatively charged DNA is present it will migrate into the agarose gel once the electric field is applied, whereas DNA in the polyplexes is retained in the pockets of the gel. Two markers are typically used. The blue dye bromophenol blue (BPB) marks the front of the migrating zone during analysis, while the DNA-intercalating dye ethidium bromide (EtBr) is used afterwards to stain the DNA (both free and polyplexed). Figure 3 left hand side 15


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summarizes the behavior of polyplexes formed in 150 mM NaCl or HBG as a function of the N/P-ratio. Results for polyplexes formed in 300 mM NaCl are shown in Figure 3 right hand side. As expected higher N/P-ratios are required for full pDNA complexation in 300 mM NaCl than in 150 mM. The trend is particular clear in case of m-PDMAEMA and can be explained by a shift in the thermodynamic equilibrium of the polyelectrolyte complex formation as a function of the salt concentration.

Figure 3. Gel retardation assay A) m-PDMAEMA B) l-PEI C1) PHMG-b-PCL12 C2) PHMG-b-PCL5 C3) PHMG-b-PCL6 1% agarose gels, N/P ratios as well as the matrices chosen for polyplex formation are indicated on top of the lanes.

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In the gels of the PHMG-b-PCLx polyplexes (x = 5 or 6), BPB fails to mark the migrating front and seems to be retained in the pockets of the gel. BPB has been shown to bind to hydrophobic sites on proteins35 and might therefore also interact with accessible hydrophobic block in the polymers. The fact that the phenomenon was observed for PHMG-b-PCLx, but not for mPDMAEMA supports our assumption that in the latter structure the hydrophobic blocks form a micellar core and are not available for interaction. When the experiments in case of PHMG-b-PCLx were repeated using a loading buffer without BPB, Figure 3 right hand side, the blue color in the pocket did indeed disappear. However, unexpectedly and in contrast to all other investigated polyplexes, it was still not possible to stain the DNA in the pockets of the gels with EtBr. The affect was particularly strong in case of polyplexes formed at higher N/P-ratios and in 300 mM NaCl as matrix. Since DNA must have been present, EtBr presumably was somehow prevented from intercalating into it. A possibility is a pronounced hydrophobic contribution to the interaction of the pDNA with PHMG-b-PCLx. Such an interaction would interfere with the hydrophobic forces stabilizing the two strands of the double helix and thereby with the site required for of intercalation.

3.3. ∆ T assay We have previously described the ∆T assay as a method to evaluate the strength of the interaction between polycation and pDNA in polyplexes26. The assay is based on the assumption that the strong electrostatic interaction between the negatively charged backbone of the individual strands of the DNA with the polycationic complexation agent affects the stability of the DNA double helix and therefore the melting temperature, Tm, of the DNA. In our previous work, lower Tmvalues always coincided with more stable polyplexes. Here, we used the ∆T assay to investigate the influence of the polymer type and the complexation matrix (150 mM NaCl, HBG) on the 17


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DNA melting temperature as a function of the N/P-ratio. As verified beforehand, in both 150 mM NaCl and HBG solution the Tm of the uncomplexed pDNA was similar and > 90 °C. From previous experience, a more or less steep decrease in Tm of the DNA was expected with increasing N/P-ratio, eventually leveling off to a plateau value at a N/P-ratio where all accessible phosphate groups of the pDNA are complexed. This behavior was observed for all polymers investigated herein, with the notable exception of m-PDMAEMA polyplexes formed in the presence of 150 mM NaCl. Average Tm-plateau values of 37°C (pDNA/l-PEI) and 27°C (pDNA/PHMG-b-PCLx, with x = 5 and 6) were found, Figure 4A, indicating a higher stability of the PHMG-b-PCLx complexes, debatably due to hydrophobic interactions modulating the electrostatic ones and thereby stabilizing the polyplexes. In consequence, conditions favoring hydrophobic interactions, such as the use of NaCl rather than HBG as matrix, strengthen this effect, Figure 4A, since the N/P-ratio required to reach the plateau Tm is lower. Apart from hydrophobic interactions, this higher stability observed for the PHMG-b-PCLx-based polyplexes might also be related to the sterically non-hindered amino-groups in PHMG-b-PCLx. pDNA binds more strongly to polycations with primary amino groups than to polycations containing secondary or tertiary one, like l-PEI and m-PDMAEMA36. The development for Tm in m-PDMAEMA polyplexes formed in HBG is similar to that seen for l-PEI and PHMG-b-PCLx polyplexes. Tm reaches a plateau of ca. 30°C at an N/P-ratio of 3, Figure 4B. m-PDMAEMA polyplexes formed in the presence of 150 mM NaCl, on the other hand, initially show a similar decrease in Tm, also reaching a minimum value of ca. 30 °C at N/P = 3. However, instead of stabilizing, Tm increases again with increasing N/P-ratio, finally reaching a value of ca. 75°C at N/P-ratios > 7.5. We hypothesize that a change in the charge density of the polycation is responsible for this unusual behavior and that in fact the charge density of the involved polycation is more relevant for the stability of the polyelectrolyte 18


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complexes than the polymer concentration, that is the total number of cationic charges available for interaction with the DNA.

Figure 4. Melting temperature of polyplexed DNA as a function of the N/P-ratio. A) PHMG-b-PCL5 (), PHMG-b-PCL6 (), l-PEI (∆), B) m-PDMAEMA (), pH values () Empty symbols: 150 mM NaCl, filled symbols: HBG, data points represent mean ± SD (n ≥ 4), lines serve as guides to the eye.

Both the pH and the ionic strength of the matrix can influence the charge density of a weak polyelectrolyte such as PDMAEMA. The number of ions additionally introduced into the matrix by increasing the N/P-ratio from 3 to 7.5 is minimal compared to the 150 mM of NaCl already 19


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present. However, the pH of an unbuffered solution, such as the aqueous 150 mM NaCl solution used here, is sensitive to the addition of even small amounts of a weak polymeric base (PDMAEMA), which in turn changes the degree of protonation of the PDMAEMA in the mixture. Compared to the hexamethylene guanidinium group in PHMG-b-PCLx, which is a strong base, but also to PEI, which is known to act itself as a buffering agent, the charge density of the PDMAEMA would thus show a pronounced dependency via the pH on the N/P-ratio. The development of the pH-values of the m-PDMAEMA polyplex solutions in NaCl and HBG is also indicated in Figure 4B. Even at high N/P-ratios the pH of the buffered HBG solution stays relatively constant and close to 6.5. The addition of increasing amounts of PDMAEMA to the unbuffered NaCl solution, however, increases the pH from 5.8 (pure pDNA) to 7.6 (N/P = 20). In consequence the degree of protonation of the PDMAEMA block is expected to change from nearly 100 % at pH 5.8 to less than 50 % at pH 7.637, even if we presume a stabilizing effect of the charged (protonated) state of PDMAEMA by the salt. The lower degree of protonation of PDMAEMA at higher N/P-ratios then results in the observed weaker binding of the pDNA, which would explain the relatively high Tm observed for m-PDMAEMA at N/P-ratios ≥ 10.

3.4. Transfection efficiency and biocompatibility Whereas m-PDMAEMA and in particular l-PEI have been extensively characterized as transfection agents before2, 38, this is not the case for PHMG-b-PCLx, which presents a new type of transfection agent, combining various features deemed to improve performance. In this context, the biodegradable PCL block was expected to improve biocompatibility. To verify this, the cytotoxicity of all transfection agents was determined by standard MTT assay (ISO 10993527). The free polymers (PHMG-b-PCL5, l-PEI, m-PDMAEMA) were tested, as they tend to be

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much more toxic than the polyplexes39, while transfection cocktails are typically prepared at N/Pratios >> 1 and therefore contain significant amounts of free polycation. The LD50 of l-PEI was 13.4 ± 0.6 µg/mL, that of m-PDMAEMA 14.8 ± 1.9 µg/mL, and that of PHMG-b-PCL5 22.9 ± 2.2 µg/mL. Whereas m-PDMAEMA thus shows a cytotoxicity comparable to l-PEI, PHMG-b-PCL5 has a two-fold higher LD50 and is therefore significantly less toxic. However, size is also known to influence cytotoxicity, with smaller molecules being less toxic than larger ones of the same kind6, 40. The lower toxicity observed for the PHMG-bPCL5 compared to the other investigated polymers could therefore simply be linked to the difference in size. The final decision on this point will have to await the synthesis of larger PHMG-b-PCLx polymers. Finally transfection efficiencies (TE) of the different polymer types were compared in HEK-293 cells. Figure 5 compiles TEs established for the three PHMG-b-PCLx (x = 5, 6, 12), mPDMAEMA, and l-PEI 48 h hours post transfection. Viabilities were also determined and remained in an acceptable range. With m-PDMAEMA and l-PEI we see a statistically significant increase in TE at similar biocompatibility when HBG rather than NaCl is used in the formation of the polyplexes. A similar effect has previously been reported for m-PDMAEMA as transfection agent for CHO-K1 and L929 cells41 and for l-PEI in COS-7 cells42. Different factors may contribute to this improvement. As discussed above, smaller polyplexes are more easily taken up by the cells3, which may improve transfection in the case of polyplexes formed in HBG, but the complex stability may also play a role, for example by facilitating pDNA release or intracellular transport.

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Figure 5. Transfection efficiencies obtained in HEK-293 cells. A) grey bars: NaCl (150 mM), white bars: HBG, B) Transfection matrix containing increasing amounts of NaCl as indicated: N/P-ratio 7.5, l-PEI: N/P-ratio 20, PHMG-b-PCLx: N/P-ratio 34 Percentages of transgene (EGFP) expressing cells were determined 48 hours after transfection. The corresponding viabilities are indicated underneath the bar. Data points represent mean ± SD (n ≥ 2), * indicates statistical significance (p 20 a.u. (PIhigh), were considered dead, cells with a PI fluorescence between 0.8 and 20 a.u. (PIlow) were presumed to be alive and to have taken up the PI through a temporarily destabilized membrane.

Figure 6. Membrane permabilization in CHO cells by the free polymers

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A) Development of the PIlow cell fraction with incubation time; grey bar: negative control (HBG), dotted bar: positive control (Digitonin), striped bar: l-PEI, black bar: m-PDMAEMA. Data points represent mean ± SD from three independent experiments. B) Histograms overlays (PIlow cell fraction); negative control: line and fill color gray, positive control: dotted line, l-PEI: dashed line, m-PDMAEMA: solid line.

As shown in Figure 6, a large fraction of the cells incubated with m-PDMAEMA rapidly became transiently PI positive (72% PIlow-cells after 2 hours). Once all external PI had been removed by the media exchange after 4 hours, the fraction of the PIlow-cells slowly returned to the background level (< 30 % after 10 h). The effect of m-PDMAEMA is thus strikingly similar to that of Digitonin (80 % PIlow-cells after 2 hours decreasing to < 50% towards the end of the incubation period). l-PEI, on the other hand, induced PIlow fluorescence more slowly in a much smaller fraction of the cells. Moreover, this fraction stayed constant for the rest of the incubation period. If the similarities seen in the Digitonin- and the m-PDMAEMA-treated cells are indicative, a significant amount of PI enters the m-PDMAEMA-treated cells via a destabilized membrane and is pumped out of the cell once the destabilizing agent is removed.

Finally CHO-K1 cells were transfected in the presence of 1µg/mL PI using all three polymer types. The percentage of the PIlow-cells, the fraction of transgene expressing cells and their distribution over low, middle and high producers, were estimated every two hours for a period of 12 hours as well as 48 hours post transfection, Figure 7. In the transfections involving the PHMG-b-PCLx block copolymers, the number of PIlow-cells was always < 10 %. Contrarily to initial hypothesis, the guanidine-rich PHMG block in these molecules thus did not destabilize the

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plasma membrane sufficiently to enable PI uptake by the viable cell fraction. Not surprisingly in view of these results, transfection efficiencies with the PHMG-b-PCLx polymers were also low in case of the CHO cells. Significant further improvement of the transfection protocol and/ or agent for CHO-K1 is required before PHMG-b-PCLx polymers can become useful tools for transfecting such cells.

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Figure 7. Transfection efficiencies and membrane permabilization in CHO-K1 cells exposed to polyplexes 28


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∆: percentage of PIlow cells; bars: percentage of EGFP-expressing cells, with black segment: low producers, light grey segment: middle producers, and dark grey segment: high producers. N/P-ratios were 20 (l-PEI), 7.5 (m-PDMAEMA), and 34 (PHMG-b-PCLx). Data points represent mean ± SD ((n ≥ 3).

When the cells were transfected using l-PEI, the number of PIlow-cells increased rapidly to 90% during the first 4h, at which point the medium was exchanged, and afterwards decreased slowly to 30% over the next 44 h. The rapid initial increase to values much higher than those observed for the free polymer, is easily explained by piggybacking of the dye onto the polyplexes. Moreover, l-PEI polyplexes have been described to attach to the cell surface, in particular in the form of large aggregates46 before entering the cell by endocytosis1. The presence of such attached but not internalized aggregates would explain the observed persisting PI staining in case of the lPEI transfected cells even after formal “removal” of the polyplexes during the media exchange. This is apparently not the case for m-PDMAEMA where the PI fluorescence is more transient and decreases rapidly after media exchange. PI was taken up at similar rates as with l-PEI, but only by 60 % of the cells. After media dilution, the fraction of PIlow-cells decreased linearly to less than 10% over the next 40 h, a value similar to that of the control cells For l-PEI and m-PDMAEMA transfected cells, the fraction of transgene expressing (“green”) cells increases almost linearly during the first 12h post transfection and thereafter seems to stabilize around 60 % in case of l-PEI, Figure 7. For m-PDMAEMA the total percentage of transgene expressing cells continued to increase and almost doubled between 12 and 48 h reaching total transfection efficiencies close to 90 % at the end of the observation period (48 h). Moreover, while for l-PEI the distribution over low, middle and high producers was nearly

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constant from the start, in case of m-PDMAEMA this distribution shifted with time almost completely to side of the high producers. Some of these observations might be linked to the above-mentioned differences in the uptake mechanisms, endocytosis for l-PEI and transient permeation of the plasma membrane for m-PDMAEMA. Endocytosis means passage through endosomal vehicles with their degrading environment. While this might accelerate / direct transport (hence the quicker built up of the number of transfected cells) and while PEI is assumed to be able to finally escape the endosomes, some DNA degradation will take place. In view of our results, cellular uptake by membrane destabilization / pore formation mechanism as proposed by us for m-PDMAEMA should has been highly effective in triggering cellular internalization, but intracellular transport may be slower. More importantly, the ability to permeabilize biological membranes does not necessarily stop at the outer cellular membrane, but could also extend to the nuclear membrane. If this were the case, a propensity for membrane destabilization would largely help to solve the hitherto unanswered question of why m-PDMAEMA, contrarily to PEI, is able to transfect non-dividing cells11.

4. Conclusions Simple polycationic transfection agents have reached a level of optimization, where a further improvement of their performance becomes difficult. The limitations of these agents, for example their inability to transfect non-dividing cells are also well established. Polymers combining both electrostatic and hydrophobic interactions show a more complex pattern of interaction with the pDNA. The size, but also the stability of the polyplexes formed by these polymers, changes in a complex manner with the pH and the ionic strength of the environment. In certain cases, mechanisms other than endocytosis may be used to pass biological membranes. For standard cell lines like HEK-293 and CHO-K1, these novel agents admittedly perform no better than the 30


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standard polycations such as l-PEI. However, they already extend the range of cells susceptible to non-viral transfection and certainly deserve further investigation and optimization of their potential.

Acknowledgements

This

work

was

founded

by

the

Upper

Franconian

Trust

(Oberfrankenstiftung, Bayreuth, Germany) grant P-Nr.: 03847 to RF and AHEM. The Authors would like to thank Agnes Mezei, Jennifer Nack, Rebekka Sprick, and Madita Wolter for performing the ∆T assay.

Conflict of interest statement The authors have declared no conflict of interest

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Biomacromolecules

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Table of content

DNA complexation using polycationic l-PEI as potential gene delivery agent is compared to polyplex formation using two types of amphiphilic diblock copolymers, one forming micelles in aqueous solution, the other not. Polyplex formation is studied as a function of the matrix composition as well as in regard to possible mechanisms of transmembrane passage and DNA delivery. In case of the micellar agent, transient membrane destabilization conceivable contributes to the DNA transfer. The presence of guanidinium groups in the other amphiphilic polymer does not lead to improved membrane penetration.

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Compaction and Transmembrane Delivery of pDNA: Differences

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