Carbosilane dendrimers with phosphonium terminal groups are low toxic non-viral transfection vectors for siRNA cell delivery

Accepted Manuscript Carbosilane dendrimers with phosphonium terminal groups are low toxic nonviral transfection vectors for siRNA cell delivery Regina Herma, Dominika Wrobel, Michaela Liegertová, Monika Müllerová, Tomá š Straš ák, Marek Maly, Alena Semerádtová, Marcel Štofik, Dietmar Appelhans, Jan Maly PII: DOI: Reference:

S0378-5173(19)30196-6 https://doi.org/10.1016/j.ijpharm.2019.03.018 IJP 18202

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

28 October 2018 8 March 2019 9 March 2019

Please cite this article as: R. Herma, D. Wrobel, M. Liegertová, M. Müllerová, T. Straš ák, M. Maly, A. Semerádtová, M. Štofik, D. Appelhans, J. Maly, Carbosilane dendrimers with phosphonium terminal groups are low toxic non-viral transfection vectors for siRNA cell delivery, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.03.018

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Title:

Carbosilane dendrimers with phosphonium terminal groups are low toxic nonviral transfection vectors for siRNA cell delivery.

Authors: Regina Herma1, Dominika Wrobel1, Michaela Liegertová1, Monika Müllerová1,2, Tomáš Strašák1,2, Marek Maly1, Alena Semerádtová1, Marcel Štofik1, Dietmar Appelhans3 and Jan Maly1#

Affiliations: 1 Faculty of Science, J.E. Purkyně University in Ústí nad Labem, 40096 Ústí nad Labem, Czech Republic 2 Institute of Chemical Process Fundamentals of the CAS, v.v.i., Prague, Czech Republic 3 Leibniz Institute of Polymer Research Dresden, Hohe Straße 6, D-01069 Dresden, Germany

#corresponding author: Jan Malý Department of Biology J.E. Purkyně University in Ústí nad Labem České mládeže 8 40096 Ústí nad Labem Czech Republic Email: [email protected] Tel: +420475283376

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Abstract Non-viral gene delivery vectors studied in the gene therapy applications are often designed with the cationic nitrogen containing groups necessary for binding and cell release of nucleic acids. Disadvantage is a relatively high toxicity which restricts the in vivo use of such nanoparticles. Here we show, that the 3rd generation carbosilane dendrimers possessing (trimethyl)phosphonium (PMe3) groups on their periphery were able to effectively deliver the functional siRNA into the cells (B14, Cricetulus griseus), release it into the cytosol and finally to achieve up to 40% gene silencing of targeted gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) with the comparable or, in some cases, even better effectivity as their ammonium counterparts. Moreover, such cationic dendrimers show relatively low in vivo toxicity as compared to their ammonium analogues when analyzed by standard Fish Embryo Test (FET) on Danio rerio in vivo model, with LD50 = 6.26 µM after 48 hours of incubation. This is more than 10-fold improvement as compared to published values for various other types of cationic dendrimers. We discuss the potential of further increase of the transfection efficiency, endosomal escape and decrease of toxicity of such non-viral vectors, based on the systematic screening of different types of substituents on central phosphonium atom.

Keywords: phosphonium carbosilane dendrimers; transfection; gene therapy; non-viral vectors; small interfering RNA; molecular dynamics

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1.

Introduction

Rapidly developing concepts of gene therapy bring great expectations in potential treatment of several fatal genetic-based diseases as are cystic fibrosis, haemophilia, various types of neurodegenerative diseases, HIV infections and cancers(Miele et al., 2012). The core of the approach lies in the specific local delivery of nucleic acids (DNA, small interfering RNA (siRNA)) in to the targeted cells to mediate the therapeutic effect on selected genes. Based on the type of nucleic acid, the genetic material must be transported either into the nucleus (DNA) or into the cytosol of the cells (siRNA). The indispensable part of the functional gene therapy concept is the availability of suitable nucleic acid carriers. Among the others, so called non-viral gene delivery vectors, as are various cationic polymers and lipids, are often studied for such purpose. Due to the presence of the positive charge they undergo an electrostatic interaction with negatively charged nucleic acids and form nanoparticle complexes (polyplexes, lipoplexes) with suitable properties for cell transfection(Dizaj et al., 2014). Such nanoparticles enable to stabilize and protect nucleic acid from nuclease digestion, stimulate the cellular uptake and final release of genetic material within the cell(Dizaj et al., 2014). Dendrimers (DDMs), a group of nearly monodispersed, highly symmetric, periodically branched polymeric nanoparticles with a near-spherical shape at higher so-called generations (G), have been systematically studied over the past decade as gene delivery vectors due to their attractive properties rivaling the other types of commonly studied polymers. Due to the precisely controlled composition of their core and shell and wealth of surface modification strategies available they possess high potential to surpass many other non-viral vectors in gene delivery applications. Concept of the gene delivery based on numerous DDMs types has been demonstrated in vitro and in vivo as well(Dufès et al., 2005; Caminade et al., 2008; Oliveira et al., 2010; Shcharbin et al., 2010; Li et al., 2016; Li et al., 2018). Despite the relatively high transfection effectivity in vitro, the toxicity issues of cationic DDMs (and cationic polymers and lipids in general) in vivo are still main barrier for their practical application in gene therapy (reviewed in(Jain et al., 2010)). This problem is commonly faced by surface engineering of DDMs with low-toxic outer shell (e.g. poly(ethylene)glycol(Malý et al., 2009; Jain et al., 2010; Zhao et al., 2010; Zhu et al., 2010; Maly et al., 2016), carbohydrates(Maly et al., 2012; Wrobel et al., 2015; Wrobel et al., 2017; Liegertová et al., 2018), amino acids(Agashe et al., 2006), acetyls(Nimesh et al., 2007), phospholipids(Liu et al., 2017) etc.), which partially shield the positively charged groups present in the inner layer of dendrimer. This may, on the other hand, reduce the stability of DDM-nucleic acid complexes (dendriplexes, DPXs) and transfection efficiency, so it compromises the final effectiveness of DPXs in gene delivery. Most of the cationic polymeric materials (including the DDMs) or lipids used in transfection studies were, up to now, based on positively charged nitrogen-containing groups including the ammonium and imidazolium cations (reviewed in(Dufès et al., 2005; Shcharbin et al., 2010)). Contrary, only few studies have been dedicated to other types of cationic groups as are e.g. phosphonium or arsenium cationic moieties(Floch et al., 2000; Guenin et al., 2000; Picquet et al., 2005; Biswas et al., 2012; Fraix et al., 2012; Hemp et al., 2012a; Hemp et al., 2012b; Ornelas-Megiatto et al., 2012; Wang et al., 2014; Strasak et al., 2017). It has been already shown, that various types of phosphonium containing compounds were less toxic than their ammonium analogs(Jurij et al., 1995; Floch et al., 2000; Guenin et al., 2000; Picquet et al., 2005; Kumar and Malhotra, 2009; Ornelas-Megiatto et al., 2012; Strasak et al., 2017). Moreover, higher transfection efficiency of the phosphonium groups was often observed either with the lipid based transfectants(Floch et al., 2000; Guenin et al., 2000; Picquet 3

et al., 2005; Fraix et al., 2012) or phosphonium modified polymers(Biswas et al., 2012; Hemp et al., 2012a; Hemp et al., 2012b; Ornelas-Megiatto et al., 2012) as were, e.g. phosphonium-containing AB diblock copolymers(Hemp et al., 2012b), styrenic homopolymers(Hemp et al., 2012a) or polyacrylate polymers(Ornelas-Megiatto et al., 2012). In spite of the interesting properties of such materials, much less effort has been focused on synthesis of phosphonium DDMs(Biswas et al., 2012; Wang et al., 2014; Strasak et al., 2017). Moreover, there is just one literature example, describing the triphenylphosphonium (TPP) conjugated poly(amidoamine) (PAMAM) DDMs use for gene delivery(Wang et al., 2014). It has been shown, that such DDMs may easily enter the cells and mitochondria due to presence of TPP group and are effective in DNA complexation and cell transfection(Wang et al., 2014). To our best knowledge, there is no study available so far dedicated to comparison of the toxicity and the transfection effectiveness of DPXs prepared from ammonium and phosphonium dendrimer analogues. The cell delivery of siRNA and resulting gene silencing effect by use of phosphonium DDMs has not been investigated as well. Based on our previous experience in the carbosilane dendrimer (CBS-DDM) synthesis and characterizations(Pedziwiatr-Werbicka et al., 2012; Strasak et al., 2012; Gomez et al., 2013; Strasak et al., 2014; Strasak et al., 2016), we have recently synthesized a series of phosphonium modified CBSDDMs(Strasak et al., 2017), tested theirs in vitro toxicity, and also interactions with model lipid bilayers(Wrobel et al., 2018). We have shown, that the in vitro toxicity of phosphonium CBS-DDMs is similar or lower as compared to ammonium CBS-DDMs depending on the type of phosphonium group(Strasak et al., 2017). Similarly, large differences in the strength and the way of their interaction with the model lipid bilayers based on the type of phosphonium group present on DDMs were also observed(Wrobel et al., 2018). Herein, we continue such investigations with comparative study of (trimethyl)phosphonium (PMe3) CBS-DDMs and their structural analogues, (trimethyl)ammonium (NMe3) CBS-DDMs, in the: (i) in vivo toxicity in developing embryos of model fish Danio rerio (FET test); (ii) their ability to complex a model siRNA; (iii) transfection efficacy of DPXs and (iv) an effectivity of resulting gene silencing in the model cell lines (B14 fibroblasts from Cricetulus griseus). Results show, that PMe3 DDMs represent a suitable alternative to ammonium-based NMe3 DDMs with comparable transfection efficiency, but significantly lower in vivo cytotoxicity. 2. Materials and Methods 2.1 Synthesis of dendrimers Ammonium and phosphonium CBS-DDMs of generation 1-3 were prepared according the previously described method(Strasak et al., 2017) presented in Scheme 1. Briefly, allyl-terminated starting compounds were hydrosilylated by (3-chloropropyl)dimethylsilane to produce products containing 3chloropropyl terminal groups which was subsequently transformed to iodopropyl by Finkelstein reaction. Finally, formation of onium salts on the periphery of DDMs was achieved by quarternisation reaction carried out in acetonitrile with slight excess of trimethylamine or trimethylphosphine. The products were isolated as white or pale brown solids. The general structure of DDMs and their computer simulation models are presented in Fig. 1.

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Scheme 1. work

Synthesis of trimethylonium salts terminated carbosilane dendrimers used in this

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Figure 1. Structure of carbosilane dendrimers. (A) General structure of CBS-DDMs. (B) Molecular models of G1-3 of NMe3 and PMe3 DDMs (adapted from (Strasak et al., 2017)). 2.2 In vivo toxicity tests on model fish Danio rerio Fish maintenance Adult Zebrafish (Danio rerio) were kept in small schools (maximum 12 fish per 25L tanks) at 26 -28 °C with a 14/10-hour (light/dark) photoperiod. Day prior to the experiment adult fish were transferred into spawning aquariums at the end of the light photoperiod. In the tank males and females were kept separated by a plastic septum. All fish were spawned at the onset of the photoperiod by removing the septum keeping both sexes apart. All eggs were collected within 30 minutes, rinsed with culture medium and checked for health state. The culture medium was prepared according to the OECD guideline(OECD). The developmental stage was verified under a binocular microscope prior to exposure to DDMs. Fish Embryo Test FET was carried using 6 concentrations (0.001, 0.01, 0.1, 1, 10, 100 μM) of tested DDMs and controls (ISO-water and 4 mg/L of dichloranilin) over a 96-hour period at 26,9 (+/-1) °C in semi-static conditions. Each treatment consisted of 24 embryos per concentration. Prior to the FET all wells in the testing plates were saturated by an overnight incubation with tested concentrations of DDMs. Before loading the embryos to the wells, all solutions were replaced by fresh, overnight aerated solutions with corresponding concentrations or controls. Collected eggs were first transferred into 12-well plates for pre-exposure to tested solutions (45 embryos per well, per 2 ml). After 2 hours of pre-exposure 24 successfully fertilised undamaged eggs with normally developing embryos were selected using stereomicroscope and transferred individually to pre-treated plastic 96-well plates (1 embryo per well, per 200 μl). All solutions in the well plates were renewed after 48 hours of exposure to minimalize the potential reduction in the compound concentration and oxygen levels. Embryos were observed and scored for survival, morphological effects or hatching success at 24, 48, 72, 96 hours post exposure (hpe) using an inverted microscope Olympus IX71. A test was classified valid, if 90% of the embryos in the negative control displayed no morphological effect while 100% of the embryos in the positive control were considered dead at end of the 96h exposure period. Main morphological endpoints in our analysis were: (1) coagulation of fertilized eggs or developing embryos; (2) lack of somite formation; (3) lack of detachment of the tail bud from the yolk sac; (4) lack of heartbeat or heart defects (oedema); (5) malformations of body parts (head and tail region) and (6) growth retardation. All the mentioned observations were used to determine the lethality (any positive outcome means the embryo was considered dead). Zebrafish FET data evaluation For each of the assessed time points a concentration dose-response curve was calculated and the LD50 values for overall morphological effect, including the 95% confidence intervals, were determined. The percentage of embryos displaying lethal or sub-lethal morphological effects (considered endpoints in our analysis) were plotted against the concentration scale. Statistical analysis was performed using GraphPad statistical software (GraphPad Software Inc.). LD50 were estimated from curve fit provided by four-parameter logistic function. The data used were binary incidences recorded for each endpoint.

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2.3 Preparation of dendriplexes DPXs were formed by mixing defined volumes of DDMs and siRNA (Silencer™ GAPDH siRNA, ThermoFisher scientific, cat. No. AM4632) and/or negative siRNA (MISSION® siRNA Universal Negative Controls, Sigma-Aldrich, cat.No SIC 001) dissolved in sterile PBS (pH = 7.4) or TAE (pH = 8.0) buffer at a defined (+/-) molar charge ratio. The mixture was vortexed and incubated for 30 min at room temperature prior to further use. Eventually, the fluorescently labeled siRNAs (as indicated further in the text) were used in some experiments. 2.4 Gel retardation electrophoresis experiments DPXs prepared at different molar charge ratios were subjected to electrophoresis through a 1.8 % agarose gel in a 1x Tris–acetate–EDTA (0.04 M Tris-acetate, 0.001 M EDTA, pH = 8.0) with ethidium bromide (0.1 – 0.5 µg/ml gel) at room temperature. Samples were electrophoresed at 80 V for 45 min and the gel was later documented under UV irradiation (Gel Logic 112, CareStream). 2.5 Dynamic light scattering The hydrodynamic diameter (Dh) and size distribution (Z-average mean) of particles were measured using dynamic light scattering (DLS) method in a Zetasizer Nano ZS instrument (Malvern Instruments, UK). The refraction factor was assumed 1.33, the wavelength of laser was 633 nm and the detection angle 173°. The samples prepared in 100 mM PBS buffer pH = 7.4 were placed into the plastic cells ZEN0040 (Malvern Instruments, UK) and measured at 25 °C. The data were analyzed using the Malvern software v7.10. 2.6 Zeta-potential measurements The particle charge measurements were conducted using a phase analysis of the light scattering with Zetasizer Nano ZS (Malvern Instrument, UK). The electrophoretic mobility of the samples in an applied electric field was measured in Malvern capillary plastic cells DTS1061 (Malvern). Samples were prepared and measured at 25°C in dH20. Six zeta potential measurements were collected for each dispersion, and the results were averaged. The zeta potential value was calculated by using the Malvern software v7.10. which works with the Helmholtz-Smoluchowski equation. 2.7 Fluorescence anisotropy Fluorescence anisotropy measurements were carried out with a FluoroMax-4 spectrofluorimeter (Horiba Scientific, France). Samples were prepared in charge ratio 1/1 to 9/1 (dendrimer(+)/siRNA(-)) with fluorescently labeled negative control siRNA (6-FAM, Sigma-Aldrich Inc.). After 30 min of incubation (37°C), samples were supplemented with the 1 x TAE buffer to a final volume of 200 µl and measured. The excitation and emission wavelengths were λ = 490 nm and λ = 517 nm, respectively. The width of the slots for both monochromators was set at 6 nm. Six independent measurements were performed of 25°C and results were averaged. The anisotropy values (r) of the samples were calculated by the fluorescence data manager program (FluorEssence software) using the following equation: r = (IVV - GIVH)/(IVV + 2GIVH)

(1)

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where IVV and IVH are the vertical and horizontal fluorescence intensities, respectively, to the vertical polarization of the excitation light beam. The factor G = IHV/IHH (grating factor) corrects the wavelength response to the polarization of the emission optics and detectors. 2.8 Nuclease protection assay Nuclease protection assay determines the protective role of DPX against the degradation by the restriction endonucleases. Assay was performed according to ref. (Shcharbin et al., 2010). NMe3/PMe3negative control siRNA DPXs were prepared at the ratio 3/1 (+/-). After 30 min. of incubation at 37°C a 0.3 ml RNase solution (10 µl/ml, Sigma-Aldrich Inc.) was added and mixture was again incubated for additional 30 min. After that, 5 µl of heparin (160 µl/ml, Sigma-Aldrich Inc.) and 7 µl of loading buffer (30 % glycerol in dH2O) was pipetted into the solution and content was gently mixed. Samples were electrophoresed at 80 V for 45 min. through a 1.8 % agarose gel in a 1x Tris–acetate–EDTA buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH = 8.0) with ethidium bromide (0.1 – 0.5 μg/ml gel) at room temperature and documented under UV irradiation (Gel Logic 112, CareStream). 2.9 Atomic force microscopy Atomic force microscopy (AFM) analysis of DDMs and DPXs was performed using an AFM Integra Probe Nanolaboratory (NT-MDT, Moscow, Russia). Dry samples were analyzed in semicontact mode with a 100 × 100 μm closed-loop scanner (scanning by sample). NMe3/PMe3-negative control siRNA DPXs were prepared from 10 nM solution of DDMs and siRNA at the 3/1 (+/-) charge ratio. 20 µl of DPX solution was pipetted on freshly cleaved mica surface. After 10 min. incubation the mica surface was gently flushed with dH2O to remove an excess of sample and dried with a stream of nitrogen. Samples were analyzed by high accuracy noncontact composite (HA_NC) ETALON silicon tip cantilevers (NTMDT, Russia) at a typical resonant frequency of 280 kHz, using a tip radius of 10 nm and a force constant of 11.5 N/m in air, temperature and humidity of the ambient environment. Samples were scanned at 0.5 - 1 Hz for the best resolution. The data were always collected at least from three different samples, with two different tips and in a minimum of eight different positions on each sample. Only representative images were selected for presentation of the results. Image analysis was performed using Scanning Probe Image Processor software (Image Metrology A/S, Hørsholm, Denmark). Raw images were corrected for tilting of the sample stage and were zero-leveled based on the dominant height value in the distribution histogram. 2.10 Cryogenic transmission electron microscopy Cryogenic transmission electron microscopy (cryo-TEM) images were recorded in Libra 120 microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). 2 µL of specimen was placed onto each side of a holey carbon TEM grid (Quantifoil R2/2, 300 mesh), blotted with filter paper and vitrified in liquid ethane at -178 °C using a Grid Plunger (Leica Microsystems GmbH, Wetzlar, Germany). Frozen grids were inspected in the TEM using a Gatan 626 (Gatan GmbH, München, Germany) cryo-holder. Images were recorded at an accelerating voltage of 120 kV using an energy filter (zero-loss imaging) while keeping the specimen at -170 °C. 2.11 Molecular modelling Computer models of dendritic structures were created using dendrimer builder, as implemented in the Materials Studio software package from BIOVIA (formerly Accelrys). The RESP technique(Bayly et al., 1993) was used for calculation of dendrimer atoms partial charges. For this charge parameterization 8

the R.E.D.-IV tools(Dupradeau et al., 2010) was used. Generalized Amber Force Field (GAFF)(Wang et al., 2004) was used for parameterization of DDMs. siRNA molecule (5'ACUUCUCCGAACGUGUCACdTdT-3´, 3´-dTdT UGAAGAGGCUUGCACAGUG-5') was created using Nucleic acid builder (NAB) as implemented in AMBER16 software(D.A. Case, 2017) and parameterized using amber force filed ff14SB or more precisely its RNA subvariant ff99bsc0_chiOL3. Molecular dynamics simulations (T = 294 K and P = 0.1 MPa, 100 ns and 150 ns long for small and big complexes, respectively), using pmemd.cuda(Gotz et al., 2012) module from the Amber16 package, were done to obtain final DPXs. In case of small (1+1) complexes, the free energy of binding was calculated using the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) methodology as implemented in the Amber16 routine MMPBSA.py(Miller et al., 2012). Please see supporting information for more details. 2.12 Cell culture The in vitro cell culture of B14 cells (Chinese Hamster, Cricetulus griseus, ATCC, CCL-14.1, Sigma-Aldrich Inc.), was maintained in complete High glucose DMEM medium with 10 % (v/v) fetal bovine serum (FBS) and 4 mM glutamine. The cells were routinely maintained on plastic tissue culture dishes (Falcon) at 37 °C in a humidified atmosphere containing 5 % CO2/95 % air (incubator). All media contained antibiotic and antimycotic agent (100 units penicillin, 0.1 mg streptomycin and 0.25 mg amphotericin B per 1 ml of medium). Cells were harvested and used in experiments after obtaining 80-90 % confluence. The number of viable cells was determined by trypan blue exclusion on a haemocytometer. 2.13 Cell transfection studies The cells B14 were seeded with serum - Opti-MEM® Medium (Gibco™) and serum-free medium - OptiMEM® I Reduced Serum Medium (Gibco™) at concentration 12500 cells/well in a 96-well plate and transfected with 10 µl of transfections reagents solutions (final volume of transfection solution was 100 µL). DPXs prepared from the 3rd generation NMe3 or PMe3 DDMs dissolved in sterile PBS (pH = 7.4) and complexed with fluorescently labeled siRNA (10 µM stock solution of BLOCK-iT™ Alexa Fluor® Red Fluorescent, Ambion™ diluted in Opti-MEM® (Gibco™) to 200 nM solution) at molar charge ratio 7/1 (+/-) providing final 10 nM solution of siRNA/well (approx. 192 nM solution of DDMs/well) were used as transfection agents. The lipoplexes prepared from siRNA of the same concentration and Lipofectamine® RNAiMAX transfection reagent diluted in Opti-MEM® Medium (Gibco™) were always used as a control. After six hours of cell incubation in the presence of transfection agents, the 100 µl DMEM + GlutaMAX™ growth medium (Gibco®) was added to the serum-free wells and cells were incubated overnight (at least 24 hours after transfection, based on type of experiment) at 37 °C in a humidified atmosphere containing 5 % CO2/95 % air. 2.14 Flow cytometry Cells were seeded in 96-well plates at the density of 1.0x105 cells/well in serum and serum-free medium. Immediately after seeding reverse transfection was performed. Cy5-labeled siRNA (Negative control Cyanin #5, Sigma-Aldrich) was incubated with CBS-DDMs for 30 min to obtain DPXs. Prepared DPXs were then added to cells. After additional six hours of incubation serum medium was added to the serum-free wells and incubated for 18 h (24 h completely) to attach cells to the plates. In parallel, samples with the same siRNA and Lipofectamine® RNAiMAX Reagent (ThermoFisher Scientific) were prepared as a control. Prior to analysis, culture medium was removed, cells were washed with PBS (pH 9

= 7.4) and trypsinized. Cells were than suspended in a focusing fluid and assayed using the flow cytometer Attune NxT Flow Cytometer (ThermoFisher Scientific, USA). Results were analyzed by Attune NxT Flow Cytometer Software and FCS Express 6 software (De Novo Software Inc.). For excitation of fluorescence red laser (637nm) with a red emission filter RL1 (656-684nm) was used. The number of cells analyzed for each sample was 1.0x104. Results are presented as an average value ± SD from at least three independent experiments. 2.15 Confocal fluorescence microscopy The B14 cells were seeded with serum - Opti-MEM® Medium (Gibco) in a custom made microchambers (10.000 cells) and transfected by DPXs (final volume of solution was 150 µL). DPXs were prepared from the 3rd generation NMe3 or PMe3 DDMs dissolved in sterile PBS (pH = 7.4) and complexed with fluorescently labeled siRNA (Alexa Fluor® 555, Sigma-Aldrich Inc.) at 7/1 (+/-) molar charge ratio. Cells were than incubated 24 hours at 37 °C in a humidified atmosphere containing 5 % CO2/95 % air. After incubation the transfected cells were gently washed by sterile PBS (pH = 7.4) and stained with Hoechst 33342 (cell nuclei, Sigma-Aldrich Inc.) and LysoTracker Green DND-26 (lysosomes, ThermoFisher Scientific). Cells were than imaged by Leica TCS SP8 confocal microscope (Leica Microsystems) with excitation wavelength 405 nm (cells nuclei), 488 nm (lysosomes) and 561 nm (DPXs) under oil immersion objective at 63x magnification. 2.16 MTT assay Mitochondrial dehydrogenase activity was determined by the MTT test. B14 cells were suspended in media in a concentration of 1 x 105 cells/ml and plated in a flat bottom 96-well plates. Plates with the cells were incubated 24 h at a 37 °C in a humidified atmosphere of 5 % CO2 to allow the adherence of the cells before the administration of DPXs. After 24 h incubation the cells were treated with solution of DPXs. After additional 24 h of incubation, a solution of MTT in PBS was added to each well. Four hours later the medium was removed, and the formazan precipitate was dissolved in DMSO for absorbance measurement at λ = 580 nm (reference λ = 700 nm). Viability is given graphically as a percent of the control values (without DPXs). 2.17 Quantitative real-time PCR analysis of siRNA gene silencing B14 cells were incubated 24/48 hours in the presence of transfectant - 10 nM GAPDH siRNA (Thermo Fisher Scientific Inc.) complexed with the Lipofectamine® RNAiMAX Reagent and/or with the 3rd generation PMe3 or NMe3 dendrimer (7/1 (+/-) charge ratio). After incubation culture medium was removed, cells were washed with PBS (pH = 7.4) and detached from the plate (37 °C for 2 min) by trypsin. Transfected cells were suspended in DMEM + GlutaMAX™ growth medium (Gibco®). RNeasy kit (Qiagen) was used to extract total RNA from the transfected cells and controls according to the manufacturer instructions. The integrity and concentration of the isolated mRNAs were measured by spectrophotometry. Residual DNA was digested using the DNA-free kit following manufacturer recommendations (Ambion). cDNA was produced using SSIII transcriptase reagents and protocol (Thermo Fisher Scientific Inc.). Expression of the target genes was analysed by quantitative Real time-PCR LC480 (Roche) using LightCycler® 480 Probes Master assay (Roche). Commercially available hydrolysis probes for TaqMan expression assay were selected to detect the expression levels of GAPDH (probe c.n. 4331182 Thermo Fisher Scientific Inc.) used as target gene for the silencing and ACTB (probe c.n. 4331182 Thermo Fisher Scientific Inc.) used for normalisation of the qPCR data. The siRNA mediated knockdown of the target transcript was evaluated using quantitative real-time PCR 10

(LC480 Roche). Data were analysed using the LightCycler 480 Instrument Software (Roche) and Graphpad software. 3. Results and discussion 3.1 In vivo toxicity tests on model fish Danio rerio (Fish Embryo Test) The in vivo toxicity of CBS-DDMs (G3 only) was tested at the concentration range between 0.001-100 μM (Fig. 2). As expected, toxic responses were more severe following higher concentrations and longer exposures (Fig. 2a). For both dendrimer types (G3-NMe3 and G3-PMe3 DDMs) exposure to 10 μM concentration was sufficient to induce 100% mortality as early as at 24hpe. The observed morphological effect levels for both types of DDMs (Fig. 2b and Fig. 3) were mainly affected by embryo coagulation or embryo malformations, pointing to possible toxic impact on the early development of zebrafish embryos. High concentrations of DDMs seem to disrupt the process of gastrulation leading to failed organogenesis. The hatching rate of unaffected embryos (Fig. 2c) did not differ significantly when compared to controls, indicating the properties of chorion were not changed in the presence of tested DDMs. LD50 values presented in Table 1 show apparently higher toxicity of the G3-NMe3 DDM when compared to G3-PMe3 DDMs (LD50 = 0.91 µM vs. 6.26 µM for G3-NMe3 DDM vs. G3-PMe3 DDM at 48 hpe). This larger difference in LD50, observed mainly during the shorter incubation periods, supports the previous observations published elsewhere(Jurij et al., 1995; Floch et al., 2000; Guenin et al., 2000; Picquet et al., 2005; Kumar and Malhotra, 2009; Ornelas-Megiatto et al., 2012) where the lower toxic influence of phosphonium groups was described as compared to their ammonium counterparts. Interestingly, such large difference was not observed in case of in vitro studies (see Table 1 and ref. (Wrobel et al., 2018)), suggesting the certain ability of the living organism to cope better to some extent with the toxic influence of phosphonium compounds as compared to ammonium ones.

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Figure 2 FET results for embryos exposed to the 3rd generation ammonium and phosphonium dendrimers. Graphs showing response of zebrafish embryos to various concentrations of (A) G3-NMe3 DDMs and (B) G3 PMe3 DDMs; a) Dose-response curves for the assessed time points; b) Morphological effects caused by the exposure to individual concentrations over the whole 96 hours period; c) Hatching rate and success at 72 and 96 hours post exposure. note: Absence of error bars means a single FET experiment with 24 embryos per concentration as individual replicates.

Dendrimer G3-NMe3 DDM (this study) G3-PMe3 DDM (this study) G3-NMe3 DDM (Strasak et al., 2017) G3-PMe3 DDM (Strasak et al., 2017)

LD50 at 48 hpe 2

0.91 µM (R = 0.97) 6.26 µM (R2 = 0.99)* n/a n/a

LD50 at 96 hpe 2

0.89 µM (R = 0.95) 1.83 µM (R2 = 0.99)* n/a n/a

EC50 (MTT assay) n/a n/a 2.29 µM 4.43 µM

Table 1 LD50 concentrations for G3-NMe3 and G3-PMe3 DDMs in zebrafish embryos calculated through interpolation of mortality across the six tested concentrations and EC50 values for the same dendrimers on B14 cell lines obtained by MTT assay (from ref. (Strasak et al., 2017)). *note: for more precise estimation of G3-PMe3 DDM LD50 more data from the 1-10µM concertation range would be needed. Based on comparison of LD50 and EC50 values, the in vivo developmental toxicity of both DDMs at 96 hpe is a little bit higher than in vitro cytotoxicity (measured after 24 hours by MTT) described in our previous work (see Table 1 and ref. (Strasak et al., 2017)). Similar results, even with much larger disproportions, frequently up to 1-2 orders of magnitude difference, were often observed by others for various DDMs types(Bodewein et al., 2016; Liegertová et al., 2018). From this point of view as well as from the comparison with the other literature data on FET dendrimer in vivo toxicity testing (see Table 2) stems, that the presented phosphonium CBS-DDMs show up to 10-fold lower in vivo toxicity (at 48 hpf) when compared to various cationic PAMAM DDMs frequently used for siRNA delivery. This may create a significant advantage in their potential use in gene delivery applications.

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Figure 3 Representative micrographs of lethal and sub-lethal endpoints of toxicity in zebrafish embryos. a-c) Embryos recorded at 24 hours post exposure: a) Untreated control; b) Embryo showing growth retardation and failed tail detachment; c) Embryo coagulation; d-f ) Embryos recorded at 48 hours post exposure; d) Untreated control; e) Pericardial oedema; g) Embryo showing severe head and tail malformation; White arrows = head region; Grey arrows = tail region; Black arrows = heart; Y = yolk sack; Scale bar = 500 μm.

Study Calienni 2017(Calienni et al., 2017) Bodewein 2016(Bodewein et al., 2016) Oliveira 2014(Oliveira et al., 2014) This study

DDM

DDM generation

Exposure (hpf)

LD50 (48 h)

PAMAM PAMAM PAMAM

4 3-5 3,4

1-48 4-96 1-48

Carbosilane

3

1-96

G4 = 0.21 µM G3-5 = 0.56 - 0.2 µM G3 = 0.26 µM G4 = 0.16 µM G3-NMe3 = 0.91 µM G3-PMe3 = 6.26 µM

Table 2 Comparison of FET results for available dendrimer in vivo toxicity studies. Note: For the comparison only LD50 values after 48h treatment were used as this value was determined in most of the dendrimer in vivo toxicity studies. 3.2 Characterization of dendriplexes 3.2.1 Gel retardation electrophoresis experiments Gel retardation electrophoresis is a relatively simple and an efficient method for charge molar ratio optimization in DPXs(Shcharbin et al., 2009). In order to determine the ability of cationic DDMs to complex with negatively charged siRNA, we have performed a series of comparative experiments, where the type (ammonium vs. phosphonium), generation of DDM (G1-3) and the charge ratio (1/5 – 7/1 (+/-)) was systematically varied (Fig. 4A). As apparent, all DDMs were able to complex with siRNA regardles of the type of peripheral modification (PMe3 vs. NMe3), with some differences between G1 and G2,3. In case of G2-3 PMe3 and NMe3 DDMs, the visible band of negatively charged free siRNA diminishes completely at ≈ 2/1 (+/-) ratio, indicating that all siRNAs are in complex with DDMs and that the overall surface charge of the complex is neutral or positive since it does not migrate in the gel towards the anode. A slightly different trends were observable in case of G1 for both dendrimer types (PMe3, NMe3). Presence of DDMs slightly slows down the migration of siRNA in the interval between 1/5 – 3/1 (+/-) charge ratio. At ≈ 4/1 (+/-) the siRNA band diminish completely. Binding of smaller G1 DDMs to siRNA thus leads to some negatively charged intermediates, which are still able to migrate in the gel.

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Figure 4 Biophysical characterization of dendriplexes – electrophoresis and DLS. (A) Gel retardation electrophoresis of dendriplexes. DPXs were prepared by mixing of G1-3 PMe3/NMe3 DDMs and siRNA in 1 x TAE buffer pH = 8.0 at 1/5 to 7/1 (+/-) molar charge ratio. The mixture was vortexed and incubated for 30 min at room temperature prior the agarose gel electrophoresis (U = 80 V, t = 45 min.). siRNA was visualized by interaction with ethidium bromide. Pure siRNA sample was runned along with DPXs as control sample. (B) Dynamic Light Scattering – size and stability of dendriplexes. DPXs were prepared by similar approach as in (A). Instead of 1 x TAE buffer, 100 mM PBS buffer pH = 7.4 was used as solvent. Hydrodynamic sizes of DPXs were measured after 30 minutes and 24 hours of incubation (room temperature) to determine the nanoparticle size and colloidal stability of solutions. All experiments were performed in triplicate and mean values ± SD are presented. 3.2.2 Dynamic light scattering The hydrodynamic sizes of DPXs and the 24 hours stability were studied by DLS with the similar experimental conditions and variables as used in gel-retardation electrophoresis experiments (Fig. 4B). Again, a similar common trends in DPX size in case of both DDMs types - PMe3 and NMe3, were 14

observed. Generally, the maximal observed Dh after 30 min. incubation time was observed for G1 DDMs (up to approx. 1.5 µm for G1 PMe3) and lower values with increasing DDM generation (max. Dh of G2 bellow 500 nm, max. Dh of G3 DPXs bellow 200 nm). Apparently, the increasing size and the valency of the DDMs have a significant inluence on final Dh of the DPXs, with achieved lower particles sizes in case of higher generation DDMs. This may suggest more organized packing of higher generation DDMs with siRNA. The G3 PMe3 and NMe3 DPX sizes (bellow 200 nm) falls within the reasonable Dh expected for transfection particles and are comparable to that reported for other types of cationic CBS-DDMs(Pedziwiatr-Werbicka et al., 2012). DPXs prepared from PMe3 DDMs were slightly larger than those prepared from NMe3 DDMs, the fact observed mainly in case of G1 and G2. This could suggest more organized or tightly packed NMe3 DPXs as is also suggested from molecular modelling studies (as discussed in Chapter 3.3). Another important differences were observable between the DPXs formed from G1 and those prepared from G2-3 (valid for both PMe3 and NMe3) as regards to relation of size of DPXs to +/- molar charge ratio. The size of G1 DPXs increased from 1/5 – 7/1 (+/-) ratio. Contrary, the size of the G2-3 DPXs increased from 1/5 typicaly to ratio 2/1 or 3/1 (+/-) and than again decreased. Taking into account the practical use of the DPXs for cell transfection, it is clear that the structural stability should be retained during the proces of cellular uptake. Therefore, the stability of DPXs in term of Dh changes was monitored after 24hours of sample incubation. As shown in Fig. 4B, an increase of the size was observable in some experimental cases as compared to Dh measured after 30 min. In the most of G2-3 DPXs the size increase is mostly present in charge ratios close to neutral or with slight excess of positive charge which indicates a lower colloidal stability of such nanoparticles. From this point of view it seems that to guarantee the size stability of DPXs during the cell transfection, the charge ratios 3/1 (+/-) or higher should be used. 3.2.3 Zeta-potential measurements Zeta potential of DPXs is an important factor which influences the ability of nanoparticles to interact with cell membrane. Therefore, we have performed a set of measurements of zeta-potential changes of colloidal DPX solution prepared from G1-3 PMe3/NMe3 DDMs and siRNA in defined (+/-) ratio (Fig. 5A). As in the previous measurements, the similar trends and and comparable zeta-potential values were observed regardless of dendrimer type (PMe3/NMe3) used. Zeta-potential of samples with G2-3 DDMs increases from aprrox. -30 mV ((+/-) charge ratio 1/5-1/3) to +40 mV ((+/-) charge ratio 3/1-7/1) beeing close to neutral at the interval between 1/2-2/1 (+/-) charge ratio. Such results well correspond with the observable changes in gel retardation electrophoresis experiments (Fig. 4A). The charge ratio interval between the minimal and maximal values of zeta-potential roughly corresponds to observed maximal hydrodynamic sizes of each DPX type (see Fig. 4B). This can be explained by lower colloidal stability of DPXs and the tendency to form larger particles. Therefore, for transfection experiments with G2-3 DDMs, the ratio 3/1 (+/-) or higher should be preferentially used to achieve a maximal stability, positive charge and lower dimensions of nanoparticles. The first generation DPXs show again a different properties. Interestingly, even in the presence of molar excess of cationic charge they reach just neutral zeta-potential values. It may suggests a different siRNA-dendrimer assembling mechanism and structure organization of DPX as compared to G2-3 DDMs. Further more, it may also explain the increasing agregation tendency (high Dh) observed by DLS at higher (up to 7/1) +/- charge ratios and deviations (with respect to G2-3 DPXs) in electrophoretic migration observed by gel retardation electrophoresis.

15

Figure 5 Biophysical characterization of dendriplexes – zeta-potential, FL anisotropy and nuclease protection assay. (A) Zeta-potential of dendriplexes. DPXs were prepared by mixing of G1-3 PMe3/NMe3 DDMs and siRNA in dH2O at 1/5 to 7/1 (+/-) molar charge ratio. The mixture was vortexed and incubated for 30 min at room temperature before measurement. Values of zeta potential are shown as a mean ± SD (n = 6). (B) Fluorescence anisotropy changes of FAM labeled siRNA. FAMlabeled siRNA was complexed in the similar way as in (A) with DDMs (PBS pH = 7.4 was used instead of dH2O as solvent) and fluorescence anisotropy of resulting sample was measured. Data are presented as ratio of sample values (r) to control (r0) value (FAM-siRNA). Values are shown as a mean ± SD (n = 6). (C) Nuclease protection assay. DPXs were prepared at 3/1 (+/-) charge ratio from all generations of ammonium and phosphonium DDMs and universal negative siRNA. Samples were subsequently treated with heparin (160 μl/ml) (sample A) or with RNase solution (10 μl/ml, 30 minutes) followed with heparin (sample B). Samples were then subjected to agarose gel electrophoresis. 3.2.4 Fluorescence anisotropy measurements FL anisotropy measurements follow the general tendencies observed by previously discussed experiments (Fig. 5B). Similar trends were again observed for 2nd and 3rd generation NMe3 and PMe3 DDMs. The value of FL anisotropy increased within the charge ratio interval 1/5 – 1/1 (+/-) and kept the the constant value within the interval 2/1-7/1 (+/-). It can be interpreted, that FAM-labelled siRNA was fully complexed with the DDMs at approx. 1/1 (+/-) molar charge ratio. A completely different tendency was measured for the 1st generations of NMe3 and PMe3 DDMs, suggesting a more complicated mechanims of interaction. High FL anisotropy value even at 1/5 (+/-) ratio means that all FAM-labelled siRNA were complexed with the DDMs. This is in agreement with the electrophoresis experiments, where a shift of siRNA band even at 1/5 (+/-) ratio was also observed as compared to pure siRNA sample. The following decrease of FL anisotropy values with increasing (+/-) ratio suggest some further reorganization of the assembly leading to change in zeta potential and size of DPXs.

16

3.2.5 Nuclease protection assay One of the important property of suitable vectors for gene delivery is an ability to protect genetic material from nuclease digestion. Therefore, a simple nuclease protection assay was performed(Shcharbin et al., 2010) with the DPXs prepared form 1st-3rd generation NMe3 and PMe3 DDMs and siRNA in the ratio 2/1 (+/-) (Fig. 5C). Heparin as polyanionic molecule can interefere with the dedriplex which results in the siRNA release. Free siRNA in the heparin treated samples can be verified by electrophoresis experiment (Fig. 5C, first line). Furthermore, the pre-treatment of DPXs with ribonuclease and subsequent release of remaining intact siRNA confirmed the protective role of all studied DPXs againts the nuclease digestion (Fig. 5C, second line). 3.2.6 Atomic force microscopy and cryogenic transmission electron microscopy analysis AFM microscopy was performed first with the solution of 3rd generation NMe3 and PMe3 DDMs and additionally with the DPXs fabricated from the same compounds and siRNA at charge ratio 3/1 (+/-) (Fig. 6A), as representative nanostructures with suitable properties for the transfection experiments. The measured size of the DDMs in Z-scale was approx. ≈ 1.1 nm which is beyond the size (diameter) calculated from molecular modelling studies (approx. ≈ 1.9 nm, (Strasak et al., 2017)) or measured as hydrodynamic diameter (approx. ≈ 2.5 nm for NMe3 and ≈ 3.0 nm for PMe3, (Wrobel et al., 2018)). The observed difference can be explained by (i) elasticity of dendrimer and it’s deformation by the AFM tip and/or (ii) by it’s deformation due to electrostatic interactions with negatively charged mica surface, as was already discussed elsewhere(Pedziwiatr-Werbicka et al., 2012; Lim et al., 2013). The AFM analysis of DPXs revealed an existence of highly anisotropic nanoparticles with predominant thickness (measured in z-scale) at about ≈ 8 nms regardles of dendrimer type used and with much larger dimensions, of almost hundreds of nms, in x-y scale. We therefore assume a well organized packing process of DDMs and siRNA, resulting in formation of thin, flat nanoparticles, preferentially growing in x-y dimensions. It has been already discussed elsewhere(Evans et al., 2003; Peng et al., 2010; Maly et al., 2012; Pedziwiatr-Werbicka et al., 2012) that self-assembled complexes of DDMs and DNA (RNA) can acquire a highly organized anisotropic arrangements as are e.g. nanofibres, nanoplates, nanorings etc. In our case, it seems that the thickness of the objects is comparable to the length of siRNA molecules used in the experiments (bellow 10 nms). We may therefore propose a preferential upright parallel orientation of siRNA in the nanostructure, stabilized with the cationic DDMs, as described in Molecular modelling section (Chapter 3.3). The observed size in x-y for these structures roughly corresponds to hydrodynamic diameter observed by DLS (max. ≈ 200 nm at 3/1 (+/-) charge ratio). It has to be mentioned here, that DLS aproximate the measured Dh to spherical particles and can not resolve the shape of nanoparticles. The cryo-TEM experiments confirmed the existence of irregulary shaped dendriplexes (Fig. 6B). Nanoparticles of up to several tens of nanometers wide (see red arrows) were commonly present. It has to be noted here, that a larger (tens of nms in x-y) flat particles are always oriented parallel to the surface of carbon film and therefore, it is not possible to observe them from side and to measure their thickness. Such results therefore support the observations performed with AFM technique. The difference in absolute x-y size observed by AFM and cryo-TEM is based on size overestimation by AFM, which is a common weakness of this technique. 3.3 Molecular modelling Molecular modelling was focused on interaction of NMe3 and PMe3 DDMs (3rd generation only) with siRNA. First, small complexes (1 siRNA + 1 dendrimer) were simulated and binding energy calculated. 17

These simulations revealed, that both dendrimer types were able to create stable complexes with siRNA and that the affinity of NMe3 DDMs to siRNA (calculated ΔG = -79.1 kcal/mol) is higher than in case of the PMe3 DDMs (calculated ΔG = -51.6 kcal/mol) - see Table SI.1 and Fig. SI.1 and Fig. SI.2 for details.

Figure 6. Atomic force microscopy analysis and molecullar modeling of dendrimers and dendriplexes. (A) Atomic force microscopy of DDMs and DPXs. G3 NMe3 and PMe3 DDMs were 18

analyzed by AFM on mica surface on air by semicontact mode. DPXs (G3 NMe3 DPX and G3 PMe3 DPX) were prepared by mixing of G3 PMe3/NMe3 DDMs and siRNA in dH2O at 3/1 (+/-) molar charge ratio. Crossections of typical objects observed by semicontact AFM on air are presented. (B) cryo-TEM images of DPXs. G3 NMe3 and PMe3 DDMs were prepared and analyzed as described in chapter 2.10. The arrows indicate DPXs that are of flat irregular shape and that are located at the edge of the circular holes in the carbon film. Sticking to the edge of the holes is common behaviour of this class of material. The other darker spots in the images are attributed to contamination (ice particles condesed from humid air). (C) Computer molecular models of DDMs and DPXs. TOP LEFT – models of NMe3 (top) and PMe3 (bottom) third generation DDMs. TOP RIGHT Initial configuration of dendriplexes (top and side view – black ribbons). DPXs are composed of 36 DDMs and 13 siRNAs and the initial configuration is identical for both DDMs. BOTTOM LEFT (blue ribbons) - NMe3 and BOTTOM RIGHT (purple ribbons) PMe3 DPXs. Top and side views are shown. The middle siRNA molecule is highlighted using green ribbons. Situation after 150 ns of Molecular Dynamics simulation in explicit water is shown. In PMe3 case, 2 DDMs were separated during the simulation and are displayed using sphere representation (cyan color for top view and orange color for side view). Secondly, much bigger DPXs were simulated to see, how the difference in DDM/siRNA interaction affects structure of bigger DDM/siRNA complexes. These DPXs were composed of 36 DDMs and 13 siRNAs. The initial configuration reflected the information about the z-size of the DPXs from AFM analysis (i.e. z-size comparable with siRNA length) and also the request for reasonably low initial potential energy of the system was taken into account as well. This initial DPX configuration was identical for both dendrimer types and is, together with the final simulated systems, shown in Fig. 6. More compact and therefore also more rigid DPX is apparent in case of NMe3 dendrimer as compared to PMe3 one. This structural difference was confirmed and quantified using RDF analyses of simulated DPXs (see Fig. SI.3-5 in Supporting Information). These results correspond with findings obtained from simulations of small dendrimer/siRNA systems (higher dendrimer/siRNA affinity in case of NMe 3 dendrimer) and are also consistent with AFM observations. Interestingly, two dendrimer dimers were observed in both DPXs – see Fig. SI.6. This rare pairing of positively charged molecules is mediated by interaction with negatively charged siRNAs and stabilized mainly thanks to high dielectric constant of water, Van der Waals as well as with hydrophobic interactions and probably is much more abundant in DPXs with high +/- ratio where it could contribute to stability of such relatively highly charged systems. 3.4 In vitro cell transfection studies In the above discussed experiments, we have shown, that both the NMe3 and PMe3 DDMs have suitable properties for complexation and stabilization of siRNA, and that the DPXs formed at higher +/charge ratio could be potentially used as gene delivery vectors due to the overall positive charge, siRNA protection, suitable size and stability. Among the others, a 3rd generation DDMs seems to fulfill most of the requirements for succesfull gene delivery. Therefore, in the further step, we have performed a series of experiments dedicated to study of: (i) the influence of +/- charge ratio on in vitro cytotoxicity of DPXs; (ii) flow cytometry and confocal fluorescence microscopy experiments to determine the cellular uptake efficiency of DPXs and (iii) qRT-PCR experiments to prove the siRNA induced gene silencing in the DPX transfected cells. 3.4.1

Cytotoxicity and cellular uptake efficiency of dendriplexes 19

To select an optimal conditions for the cell transfection, initial experiments were dedicated to in vitro MTT cytotoxicity assay of DPXs formed from 3rd generation of NMe3 and PMe3 DDMs and negative control siRNA on modell cell line B14 (Cricetulus griseus, ATCC, CCL-14.1) at molar charge ratio 3/1, 5/1, 7/1 and 9/1 (+/-) (final tested siRNA concentration in the solution was 10 nM). Cytotoxicity of DPXs was compared to lipoplex formed from siRNA of the same concentration and lipofectamine (conditions of use as recommended by manufacturer) (Fig. 7A). At set experimental conditions, very low cytotoxicities of DPXs, comparable to lipoplex, were observed (cell viability ≈ 80-100% as compared to control) up to charge ratio 7/1 (differences between 3/1, 5/1 and 7/1 were statistically non-significant (p ≥ 0.05) for both DDMs). The 9/1 experimental variant was already more toxic (cell viability bellow 80%) than the rest of the DPXs (p < 0.05). Therefore, to maximize the probability of suscesfull transfection (more positive charge, minimum cytotoxicity), the charge ratio 7/1 (+/-) was selected for the next experiments. Transfection experiments were performed with the DPXs composed of the 3rd generation NMe3 and PMe3 DDMs and fluorescently labelled GAPDH siRNA at 7/1 (+/-) charge ratio and with the lipoplexes prepared from the lipofectamine and the same siRNA as a reference (Fig. 7B,C). Transfections were performed at two different experimental conditions: (i) in the presence of serum and (ii) in the cultivation media lacking the serum (as described in Chapter 2.12). The cellular uptake efficiency was evaluated after 24 hours by flow cytometry (Fig. 7B,C). At set conditions, both DPXs were able to deliver the siRNA into most of the cells, but the efficiency of the process was dependent on the type of the dendrimer (NMe3 vs. PMe3 DDM) and the type of cell culture media. The highest transfection was observed in serum-free media. At such conditions, the lipofectamine was approx. 3 times more effective than G3 PMe3 DPX and in similar manner, the G3 PMe3 DPX was approx. three times more effective than G3 NMe3 DPX. Contrary, much lower differences were observed in media with the presence of the serum. Here, the efficiency of siRNA delivery was comparable in case of G3 PMe3 DPX and lipofectamine (p ≥ 0.05) and both transfectants were also more sucessfull as compared to G3 NMe3 DPX (p < 0.001) (see Fig. 7C). Our results confirmed that both types of DPXs were able to deliver the siRNA into the cell and that, in accordance with the other published results(Hemp et al., 2012b; Ornelas-Megiatto et al., 2012), the phosphonium decorated DDMs were slightly more efficient in such process than the ammonium structural analoques. Another interesting point is the large difference in the transfection efficiency observed for G3 PMe3 DPX and minimal effect in case of G3 NMe3 DPX, observed in the presence/absence of serum in culture media. It was already widely discused elsewhere (Lynch and Dawson, 2008; Karmali and Simberg, 2011; Corbo et al., 2016; Wrobel et al., 2017), that so-called protein corrona or nanoparticle corrona (based on structural arrangement and mutual size difference of nanoparticles/proteins) is formed due to interaction of nanoparticles and proteins present in the blood serum. The properties of the resulting nanostructures are quite different from the original nanoparticles with implications to their cytotoxicity, drug delivery and clearence(Karmali and Simberg, 2011; Corbo et al., 2016). As a result, the formation of protein corona may negatively influence the interaction of G3 PMe3 DPX nanoparticles with the cell surface and as a consequence, it may decrease their’s cellular uptake. Since the cellular uptake of G3 NMe3 DPX is mostly independent on presence of serum, we may hypothesize that the different type of protein corrona (as compared to G3 PMe3 DPX) is formed which has not large effect on the uptake mechanism. This observed difference should be a matter of further research focused on DDMs-serum protein interaction studies. Confocal microscopy images acquired at 24h/48h post-transfection period confirmed that G3 PMe3 DPXs were efficiently uptake by the B14 cells, but large portion of them remained entrapped in 20

acidic late endosomes/lysosomes (Fig. 8). This may explain the disproportion between efficient cellular uptake of these nanoparticles and only moderate effectivity in GAPDH gene silencing as compared to Lipofectamine® RNAiMAX reagent (discussed in chapter 3.4.2). Fig. 8 shows a representative images of B14 cells labeled with Hoechst 33342 (cell nuclei, blue), LysoTracker Green DND-26 (lysosomes/late endosomes, green) and dendriplexes (fluorescent Alexa Fluor® 555 labeled siRNA, orange). It maybe observed, that predominant co-localization of orange and green colours (DPXs and lysosomes/acidic late endosomes - visible as yellow colour) is present along with the diffused orange (released siRNA/DPXs) and isolated green vesicules.

Figure 7 Cellular uptake of dendriplexes and GAPDH gene silencing. (A) MTT cell viability assay. B14 cells were incubated 24 hours in the presence of DPXs formed from 3rd generation PMe3 or NMe3 DDM (Lipofectamine® RNAiMAX Reagent as reference) and negative control siRNA (10 nM siRNA in final solution). Molar ratio of positive/negative charges of the DDM and siRNA in DPX was set to 3/1, 5/1, 7/1, 9/1 (+/-). Cell viability is expressed as the mean value (mean ± SD (n = 3)) compared to control sample (untreated cells). Unpaired t-test was used to quantify the statistically significant differences between DPXs and lipofectamine cell viability. Values p < 0.05 were supposed to be statistically significant (*), values p ≥ 0.05 non-significant (ns). (B, C) Flow cytometry analysis of cellular uptake of DPXs. a) An example of typical FL intensity histogram plots, b) Graph of median fluorescence 21

intensity. The DPXs were prepared from 3rd generation PMe3 or NMe3 DDMs (Lipofectamine® RNAiMAX Reagent as reference) and fluorescently labeled GAPDH siRNA at 7/1 (+/-) molar ratio (10 nM siRNA in final solution). Data were collected after 24 hours of incubation with transfectant. Values represents the mean of four independent experimental replicates ± SD (n=4). One-way ANOVA followed with Tukey-Kramer multiply comparison test were used to quantify the p values between each sample (statistically significant p < 0,001 (***), statistically non-significant p ≥ 0.05 (ns)). (D) GAPDH gene silencing. B14 cells were incubated 24/48 hours in the presence of transfectant (negative control/GAPDH siRNA complexed with Lipofectamine® RNAiMAX Reagent and 3rd generation PMe3 or NMe3 dendrimer (7/1 (+/-) charge ratio), 10 nM siRNA in final solution). GAPDH expression knockdown is presented as the relative remaining expression of GAPDH in samples transfected with GAPDH siRNA as compared to the control samples transfected with negative siRNA (expression of GAPDH in control is set to 1). ACTB probe was used for normalisation of the qPCR data. One-way ANOVA followed with Tukey-Kramer multiply comparison test were used to quantify the p values between each sample (statistically significant p < 0,001 (***), p < 0.05 (*) and statistically non-significant p ≥ 0.05 (ns)). Data are presented as mean ± SD (n = 6).

Figure 8 Confocal fluorescence microscopy images of transfected cells. (A) Confocal and bright-field images of B14 cells transfected with G3 PMe3 DPXs and imaged after 24 hours of incubation: a) cell nuclei coloured by Hoechst 33342; b) bright-field image of the cells; c) LysoTracker Green DND-26 labeled lysosomes/acidic late endosomes; d) internalized DPXs formed from G3 PMe3 and fluorescently labeled siRNA. (B) Overlay of all channels. 3.4.2 GAPDH gene silencing To be biologically active, the siRNA carried by the non-viral delivery system has to be delivered into the cytoplasma of cells, released and further processed by the relevant enzyme machinery. Generally, there are several different pathways, how the non-viral vectors can be internalized by the cells, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, clathrin/caveolin independent endocytosis, macropinocytosis and phagocytosis(Selby et al., 2017). Important moment 22

of the whole process is the release of the non-viral vector from the endosome, so-called endosomal escape(Vermeulen et al., 2018). The mechanisms of this process are still not very well understood, except the “proton sponge” effect described for macromolecule vectors possesing the protonisable functional groups (e.g. primary amines)(Boussif et al., 1995; Zhou et al., 2006). Therefore, we performed another experiments, focused on the study of the biologically functional siRNA molecules presence within the transfected cells, i.e. to prove a gene silencing effect. We selected a house-keeping gene for gene silencing coding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme along with the another house-keeping gene coding beta-actin (ACTB gene) as a control. The knockdown was followed by qRT-PCR analysis (Fig. 7D) at 24 and 48 hours postransfection in B14 cell line. The similar DPXs were analyzed, as in previous experiments, i.e. the G3 PMe3 and G3 NMe3 DPXs formed at 7/1 (+/-) charge ratio and lipoplexes prepared from lipofectamine as a reference. Again, a media with the serum and without the serum were tested. As a result, we observed the knock-down at all experimental variants of DPXs (≈ 0.45 – 0.80 normalized GAPDH expresion), which was a slightly less efficient process (with the exception of G3 PMe3 DPXs after 48h without serum) than that evoked by lipofectamine (≈ 0.3 – 0.4 normalized GAPDH expression). This is somewhat to be expected, since the lipofectamine was mostly more effective in cellular uptake as revealed by flow cytometry experiments. The observed knock-down for both types of DPXs in serum media was within the interval ≈ 0.6 – 0.8 (of normalized GAPDH expression) after 24 and 48 hours posttransfection, suggesting that the both DPXs were, within the observed time window, able to release functional siRNA in to the cytosol. Slightly higher knock-down efficiency of G3 PMe3 DPXs over G3 NMe3 DPXs at 24 hours was observed. The different trends could be observed in case of serum-free media. Here the knock-down efficiency slightly increased over time where the gene silencing effect achieved after 48 hours was higher than after 24 hours. Moreover, the knock-down achieved in serum-free media after 48 hours (≈ 0.4 – 0.6 GAPDH normalized expression) was significantly higher than that in serum media. Our results support the published data(Weber et al., 2008) on siRNA GAPDH knock-down of HIV-infected lymphocytes performed with other types of cationic CBS-DDMs having quaternized amonium groups on their‘s periphery (2nd generation DDMs). Authors achived up to 40 % GAPDH knockdown, when using DPXs with final 500 nM solution of siRNA. Here we have to point out, that we reached a similar knock-out efficiency by using only 10 nM siRNA solutions, that means also with much lower dendrimer concentrations (approx. 192 nM), which were practically non-toxic (at least in the case of PMe3 DDM) even to in vivo model Danio rerio. We may conclude, that both DDMs were able, at set experimental conditions, to deliver the functional siRNA into the cytosol of transfected cells and to evoke a siRNA gene silencing of targeted GAPDH gene within the 24-48 hours posttransfection period. Despite a slightly more efficient cellular uptake of G3 PMe3 DPX as compared to G3 NMe3 DPX, the final knock-down effect was comparable. We did not perform a detailed investigation of mechanisms of cellular uptake and endosomal escape of both DPXs. Due to absence of protonisable functional group, the proton sponge mechanism of endosomal escape is hardly possible. As was already discussed elsewhere(Hemp et al., 2012a), the other type of release mechanisms, yet not known, could be expected for ammonium/phosphonium functionalized polymers. Membrane disruption and pore formation due to direct interaction of cationic DDMs with the lipid membranes surrounding the endosomes are one of the posibilities(Selby et al., 2017). Recently, we have studied the interaction of five different phosphonium CBS-DDMs and ammonium CBS-DDM with the neutral as well as negatively charged liposomes(Wrobel et al., 2018). All DDMs showed interactions as well as destabilization of the lipid membranes even at low concentrations. Among the other types, DDMs with phenyl (PPh3) and methoxyphenyl P(MeOPh)3 23

phosphonium peripheral functional groups interacted much strongly and significantly changed the properties of liposomes. It was also shown by others(Ornelas-Megiatto et al., 2012), that the nature of the alkyl substituents on the phosphonium cations have an important influence on the transfection efficiency and toxicity of the polyplexes. Therefore, we suggest that the relatively low efficient endosomal escape (as compared to lipofectamine) could be improved by using another types of phosphonium groups (e.g. PPh3 and P(MeOPh)3) for CBS-DDM modification. Such experiments will be a matter of our next manuscript. Nevertheless, our experiments proved that the 3rd generation phosphonium CBS-DDMs have a potential to be used as an non-viral vectors in gene delivery with the same or even better performance, as the ammonium ones. The ability of phosphonium DDMs to induce gene silencing in complex serum media and a relatively low in vivo cytotoxicity as compared to the ammonium DDMs as well as to other types of cationic DDMs, open the space for further investigations directed to in vivo applications. However, more detailed/complex experiments need to be done using various human cell lines to inspect/verify the ability of DDMs to transfect them and to induce an effective gene silencing of e.g. selected oncogenes. 4 Conclusions Most of the cationic polymeric materials or lipids used in transfection studies were, up to now, based on positively charged nitrogen-containing groups including the ammonium and imidazolium cations. Herein we show, that the 3rd generation CBS-DDMs, possessing quaternized cationic (trimethyl)phosphonium (PMe3) peripheral functional groups, are less toxic than their (trimethyl)ammonium (NMe3) counterparts, but show up similar transfection and gene silencing effectivity. The LD50 at 48 hpf analysed by FET test on in vivo model Danio rerio was 6.26 µM for G3PMe3 DDMs, which was a significantly higher value as compared to G3-NMe3 DDMs (LD50 = 0.91 µM at 48h hpf) and as analysed for cationic PAMAM DDMs published elsewhere (more than 10-fold difference)(Oliveira et al., 2014; Bodewein et al., 2016; Calienni et al., 2017). PMe3 DDMs of 1-3rd generation were able to form DPXs with siRNA with the quite similar characteristics, as their ammonium analogues. PMe3 DPXs were successfully prepared from 1/5 – 7/1 (+/-) charge ratio and characterized by gel retardation electrophoresis, dynamic light scattering, zeta-potential and fluorescence anisotropy measurements. DPXs of 3/1-7/1 (+/-) charge ratio prepared from 2nd and 3rd generation PMe3 DDMs showed suitable properties (overall positive charge, size bellow 200 nm, substantial stability during 24h period) for transfection studies. Nuclease protection assay confirmed the stabilization effect of DPXs towards the nuclease digestion of siRNA. Atomic force microscopy and cryo-TEM analysis of the 3rd generation DPXs revealed a self-assembled, highly anisotropic nanostructures in the form of thin plates with z size ≈ 8 nms, which is roughly the length of siRNA molecules, and x-y sizes of several tens of nms. A structural arrangement of self-assembled DPXs nanostructure was proposed and studied by molecular modelling approaches. Theoretical studies revealed a stronger interaction of NMe3 DDMs with siRNA (ΔG = -79.1 kcal/mol) as compared to PMe3 DDMs (ΔG = -51.6 kcal/mol) and confirmed the stability of proposed DPXs nanostructures. Due to stronger interaction, the modelled final NMe3 DPXs nanostructures were more tightly packed than PMe3 DPXs, as confirmed by RDF analyses. Both DPXs types were practically non-toxic up to 7/1 (+/-) charge ratio (10nM negative control siRNA) as documented by MTT test on B14 cell lines. Cellular uptake studies performed in serum containing and serum free media revealed DPXs (7/1 (+/-) charge ratio, 10nM siRNA) internalization and an efficient transfection of B14 cells. Better transfection performance of PMe3 DPXs was observed, with comparable efficiency to lipofectamine transfectant in 24

serum media. Larger indicated differences in transfection efficiency of individual DPXs types in serum and serum-free media could be probably ascribed to formation of different types of protein corona on both DPXs. Confocal microscopy co-localization experiments revealed the entrapment of DPXs in acidic late endosomes/lysosomes and relatively low endosomal escape efficiency. On the other hand, PMe3 DPXs were still able to release sufficient amount of functional siRNA into the cytosol and evoke GAPDH gene silencing within 24-48 hours post transfection period (up to 40% inhibition after 24 hours in serum media). We may conclude, that PMe3 CBS-DDMs represent a suitable alternative to NMe3 CBS-DDMs in gene delivery applications with similar gene silencing efficiency, but lower in vivo cytotoxicity. We also suggest, that there is a large potential for additional improvement of both characteristics by the systematic screening of various types of substituents on central phosphonium atom. This will be a matter of our further research. Supporting information Supporting information is available. Acknowledgements The authors acknowledge the Czech Science Foundation project No. 15-05903S, Internal Grant Agency of UJEP [Project No. UJEP-SGS-2017-53-002-3], ERDF/ESF project "UniQSurf - Centre of biointerfaces and hybrid functional materials" (No. CZ.02.1.01/0.0/0.0/17_048/0007411) and the assistance provided by the Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2015073. We also thank Dr. Petr Formanek (Leibniz Institute of Polymer Research Dresden, Germany) for his kind assistance in cryo-TEM experiments.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: 28

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