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Efficient receptor mediated siRNA delivery in vitro by folic acid targeted pentablock copolymer-based micelleplexes Roman Lehner, Kegang Liu, Xueya Wang, and Patrick Hunziker Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00851 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 5, 2017

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Efficient receptor mediated siRNA delivery in vitro by folic acid targeted pentablock copolymer-based micelleplexes Roman Lehner,†,‡ Kegang Liu, †,‡ Xueya Wang, † Patrick Hunziker*,†,¶ †

Nanomedicine Research Lab CLINAM, University Hospital Basel, University of Basel,

Bernoullistrasse 20, Basel, CH-4056, Switzerland. ¶

CLINAM Foundation for Clinical Nanomedicine, Alemannengasse 12, Basel, CH-4016,

Switzerland. E-mail address: [email protected]; Tel: +41 61 26 55 581 or +41 61 26 52 525. ‡

These authors contributed equally to this work.

KEYWORDS: Polymerization, block copolymer, nanoparticles, siRNA delivery, active targeting, serum stability ABSTRACT: Novel, biocompatible polyplexes, based on the combination of cationic pentablock copolymers with folic acid functionalized copolymers were designed and developed for target-specific siRNA delivery. The resulting micelleplexes spontaneously formed polymeric micelles with a hydrophobic core surrounded directly by a cationic poly-2-(4-azidobutyl)oxazole (PABOXA) and subsequently shielded by hydrophilic poly-2-methyl-oxazole (PMOXA)

1 Environment ACS Paragon Plus

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layer. The described micelleplexes form highly stable particles even in complete serum after 24 h compared with the highly cationic polymer PEI, which show aggregate formation in serum containing buffer solution. Targeted siRNA delivery and gene knockdown could be shown using Green Fluorescent Protein (GFP) expressing HeLa cells, resulting in ~ 31% and ~ 8% suppression of the expression of GFP for targeted and non-targeted micelleplexes, respectively. Comparison studies of folic-receptor positive HeLa cells with normal folic-receptor-negative HEK293 cells revealed involvement of receptor mediated cellular uptake of fluorescently labeled siRNA. The new designed nanocarrier showed no cytotoxicity, having a potential application. The presented concept of shielding a nucleic-acid complexing cationic chains with a stealth layer and combining it with receptor ligand overcomes typical problems with undesired protein and cell interactions in delivery of nucleic acids using polymeric systems, opening new doors for application if RNA inhibition in the organism. 1. Introduction Since the discovery of RNA interference (RNAi) there has been a considerable interest in the field of biology and medicine due to its large potential for personalized treatment of several human diseases.1, 2 The potential to selectively interfere with, and stop the development of genedependent diseases turns siRNA into a highly promising therapeutic agent to suppress cancer growth. However, naked siRNA has a very short half-life when injected to the systemic circulation because of rapid enzymatic degradation and clearance by kidney or liver due to its small molecular size, in average 21 base pairs and a weight of about 13.3 kDa versus a molecular renal cut-off of 5-70 kDa for glomerular filtration.3-5 Naked siRNA molecules can also cause toxic effects by stimulation of the immune response, and may have off-target effects and saturation of the RNAi machinery.6


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Due to their polyanionic characteristics, siRNA molecules are not able to spontaneously cross the cell membrane and thus require a delivery platform, which allows cellular uptake as well as cytosolic release for efficient gene knockdown. In regard to this given fact, numerous non-viral delivery platforms have been developed because of their low immunogenic effects, tunable size and targeting properties such as cationic lipids, polymers, dendrimers and inorganic nanoparticles.7 However, a major drawback of non-viral delivery platforms is still their cationic nature. The positive charge of the delivery platforms is relevant to complex negatively charged siRNA molecules and may contribute to endosome disruption but positive charged nanoparticles often show increased cytotoxicity, and cellular uptake may be altered by unspecific binding and binding to serum proteins, which inhibits cellular uptake of the particles.8-10 To improve biodistribution, specificity of cellular uptake and to actively target the cell of interest, ligands can be incorporated that specifically bind to receptors on target cells to induce receptor-mediated interaction.11 Folic acid (FA) has been shown to be a favorable ligand to increase cellular uptake via receptor mediated endocytosis by SKOV-3, MCF-7 and KB cells using polymer based delivery systems.12-14 Folate receptors are up-regulated in more than 90% of ovarian carcinomas and expressed at higher levels in lung, brain, kidney and breast carcinoma, whereas in most normal tissue it occurs at very low levels.15 Promising candidates for in vivo therapeutic use are carrier systems composed of poly(2methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyl-oxazoline),

PMOXA-PDMS-

PMOXA, due to their inherent biocompatibility documented by Brinkhuis et al.,16 enabling the design of synthetic organelles with intelligent sensor-effector functionality.17,18 These copolymers form highly stable micelles or closed vesicles with a controlled diameter through self-assembly in aqueous solution.19 In addition, they do not show cytotoxicity, have only limited


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nonspecific plasma protein binding due to their hydrophilic outer layer and avoid detection by the immune system. Using PMOXA-PDMS-PMOXA polymer based nanomaterials for drug delivery,17,

18

imaging20 and photo dynamic therapy (PDT)21 has been reported. However, limited information is available about its application of gene delivery, in particular for receptor-targeted delivery. Progressing from reported PMOXA-PDMS-PMOXA block copolymers, we report here for the first time the design and development of new pentablock copolymers, further refining the PMOXA-PDMS-PMOXA structure, and document its potential for targeted gene delivery. ThesiRNA drug delivery system reported here was achieved by combining the cationic pentablock copolymers with folic acid functionalized copolymers for target specific interactions. The synthesized pentablock copolymer based siRNA delivery system achieved colloidal stability in complete serum, led to siRNA condensation, proved successful for targeted delivery, endosomal escape and RNA inhibition. The neutral overall surface charge characterizing this system is thought to be important in preventing possible interactions with negatively charged serum proteins and limit unspecific cell binding. 2. Material and Methods 2.1. Synthesis of block copolymers All the substances were purchased from Sigma-Aldrich (St. Gallen, Switzerland) and abcr (Karlsruhe, Germany) and were used as received unless otherwise stated. 2-methyl-2-oxazoline, chloroform and acetonitrile were dried by refluxing over CaH2 under dry argon atmosphere and subsequent distillation prior to use. 1H-NMR spectra were recorder with a Bruker DPX-400 spectrometer (Bruker, Switzerland). Deuterated chloroform (99.8% D) and Deuterated methanol (99.8%) were used as solvent. Spectra were analysed with MestReNova 7.0.3 software


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(Mestrelab Research SL, Spain) and calibrated using solvent signals (CDCl3, 7.26ppm; CD3OD, 3.31ppm; D2O, 4.79ppm). Infrared (IR) spectra were recorded on a Perkin Elmer Spectrum One FTIR spectrometer as KBr pellets or films on NaCl plates. Most characteristic absorption bands of the spectrum were given in cm−1.The molecular weights of the polymers were obtained from 1

H-NMR calculation. 2.1.1. Synthesis of copolymer 2 To a solution of Poly(dimethylsiloxane), bis(hydroxyalkyl) terminated (HO-PDMS-OH) (5.6g,

1mmol) in toluene (25mL) was added 2,6-lutidine (0.35mL, 3mmol), followed by addition of a solution of trifluoromethanesulfonic anhydride (0.4mL, 2.4mmol) in hexane (2.5mL) dropwise in ice bath under argon. The reaction mixture was stirred at same temperature for 3h. Then solvent was removed under vacuum to give an active intermediate as yellowish oil. The oil was dissolved in a mixed solvent of chloroform (15mL) and acetonitrile (20mL). First monomer 2-(4azidobutyl)-oxazole22 (1.03g, 6.1mmol) was added at room temperature (r.t.). The reaction mixture was heated and stirred at 60oC for 43h. Then it was cooled to r.t. and second monomer 2-methyl-oxazoline (1.7mL, 20mmol) was added. The reaction mixture was heated to 60oC again and stirred at same temperature for 48h. Then it was quenched with a solution of triethylamine in water at r.t. Solvent was removed by evaporation to afford a residue. The residue was purified by ultra-filtration with cellulose regenerated membrane (a cut off at 5000 dalton) using ethanol/water (80:20, v/v, at least for four times. In the first time, a 5 ml of saturated NaHCO3 solution was added) as an eluent to collect 6.5g compound 2 as slightly yellowish solid after twice washings with hexane. Yield: 78%, 1HNMR (400MHz, CDCl3) δ 3.30-3.75 (m, 128H), 2.30-2.50 (m, 12H), 2.05-2.21 (m, 60H), 1.50-1.75 (m, 28H), 0.50 (m, 4H), 0.02-0.09 (m, 444H) ppm.


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FTIR (film): 3419, 2963, 2098, 1634, 1486, 1368, 1094, 799 cm−1. 2.1. 2. Synthesis of copolymer 3 To a solution of compound 2 (840mg, 0.1mmol) in methanol (5mL) was added trimethylamine (0.56mg, 4.0mmol) at r.t., followed by addition of 1, 3-propandithiol (0.40mL, 4.0mmol). The reaction mixture was stirred at r.t. for 12h. Solvent was removed under vacuum to afford a residue. The residue was purified by ultra-filtration with cellulose regenerated membrane (a cut off at 5000 dalton) using ethanol/water (80:20, v/v, at least for four times) as eluent to produce 560mg compound 3 as a slightly yellowish solid. Yield: 67%. 1HNMR (400MHz, CD3OD) δ 3.38-3.79 (m, 116H), 2.68 (m, 12H), 2.30-2.53 (m, 12H), 2.05-2.15 (m, 60H), 1.45-1.70 (m, 28H), 0.57 (m, 4H), 0.03-0.17 (m, 444H) ppm. FTIR (film): 3436, 2963, 2098, 1634, 1486, 1423, 1094, 800cm−1. 2.1.3. Synthesis of compound 4 To a solution of Poly(dimethylsiloxane), bis(hydroxyalkyl) terminated HO-PDMS-OH (5.6g, 1mmol) in toluene (25mL) was added 2,6-lutidine (0.35mL, 3mmol), followed by addition of a solution of trifluoromethanesulfonic anhydride (0.4mL, 2.4mmol) in hexane (2.5mL) dropwise in ice bath under argon. The reaction mixture was stirred at same temperature for 3h. Then solvent was removed under vacuum to give an active intermediate as yellowish oil. The oil was dissolved in a mixed solvent of chloroform (15mL) and acetonitrile (20mL). Monomer 2-methyloxazoline (3.4mL, 20mmol) was added at r.t. The reaction mixture was heated to 60oC again and stirred at some temperature for 48h. Then it was quenched with sodium azide (2.6g, 40mmol) at r.t. Solvent was removed by evaporation to afford a residue. The residue was purified by ultrafiltration with cellulose regenerated membrane (a cut off at 5000 dalton) using ethanol/water (80:20, v/v, at least for four times. In the first time, a 5 ml of saturated NaHCO3 solution was


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added) as an eluent to produce 6.5g compound 4 as slightly yellowish solid after twice washings with Hexane. Yield: 72%, 1HNMR (400MHz, CDCl3) δ 3.25-3.70 (m, 160H), 2.05-2.21 (m, 120H), 1.56 (m, 4H), 0.50 (m, 4H), -0.02-0.20 (m, 444H) ppm. FTIR (film): 2963, 2104, 1634, 1485, 1423, 1367, 1094, 1022, 801cm−1. 2.1.4. Synthesis of folic acid conjugated copolymer 6 To a solution of polymer 4 (880mg, 0.1mmol) in DMSO (10mL, degassed) was added compound 5 (148mg, 0.3mmol), followed by addition of solid CuSO4.5H2O (9.3mg, 0.038mmol) and sodium ascorbate (19.8mg, 0.1mmol) successively at r.t. under argon. The reaction mixture was stirred at r.t. for 24h. An ethylenediaminetetraacetic acid (EDTA) solution (2ml, 0.01M) was added. The mixture was directly purified by ultra-filtration with cellulose regenerated membrane (a cut off at 5000 dalton) using ethanol/water (80:20, v/v, at least for four times) as eluent to produce compound 6 (891mg, folic acid: 20% loading amount) as slightly yellowish solid. Yield: 91%. 1HNMR (400MHz, CD3OD) δ 8.72 (m, 2H), 7.90 (m, 2H), 7.71 (m, 4H), 6,74 (m, 4H), 4.20-4.90 (m, 14H), 3.35-3.70 (m, 160H), 1.90-2.25 (m, 128H), 1.61 (m, 4H), 0.58 (m, 4H), 0.05-0.14 (m, 444H) ppm. FTIR (film): 3445, 3054, 2963, 2306, 1645, 1634, 1480, 1423, 1367, 1095, 1022, 804, 741, 704cm−1. 2.2. Preparation of siRNA loaded pentablock based micelles The siRNA loaded FA-PMOXA-PDMS/PMOXA-PABOXA-PDMS based micelles were prepared by aqueous self-assembly. Briefly, the polymers were dissolved in 200 µl ethanol under permanent stirring to give 2.5 % (w/v) solutions. The two polymers were mixed accordingly to the N/P ratio considering that the FA-PMOXA-PDMS/PMOXA-PABOXA-PDMS mixture has a


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0.5 mol% FA-PMOXA-PDMS content. The desired amount of this ethanolic solutions were subsequently added to 100 µl of 10 mM PBS buffer containing 40 pmol of siRNA and the mixture was vortexed for 2 min followed by gentle stirring for 1 h at room temperature. 2.3. Determination of siRNA loading capacity of pentablock based micelleplexes The condensing ability of the pentablock based micelles was examined by agarose gel electrophoresis. The siRNA/copolymer polyplexes (FA-PMOXA-PDMS/PMOXA-PABOXAPDMS) were self-assembled at different charge ratios (N/P) 0.5, 1, 2, 5, 10, 15, 30 and naked siRNA was used as a control. For electrophoretic separation, samples were loaded on a 1 % agarose gel and visualized with ethidium bromide (0.2µg/ml). The retardation of siRNA mobility was detected via irradiation with UV light. 2.4. Particle size and surface charge The particle size (diameter, nm) and the zeta potential of the nanoparticles were examined using a zeta sizer (Nano ZS, Malvern Instruments Ltd., Malvern, UK) by dynamic light scattering (DLS) and electrophoretic mobility respectively. All samples were measured in 10 mM PBS buffer pH 7.4 at 25 °C. Data were given as mean ± standard deviation (SD) based on three independent measurements. Transmission electron microscopy (TEM) images have been taken to confirm the size of the complexes. 2.5. Transmission electron microscopy Transmission electron microscopy (TEM) was employed as imaging technique to visualize the size of the pentablock based micelleplexes as well as to investigate aggregation formation of the micelleplexes and polyethylenimine in serum containing buffer solution. Samples were placed on a copper grid covered with a nitroglycerin film coated with carbon. A staining agent was added (2 % uranyl acetate).


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2.6. Preparation of PEI solution Linear 25 kDa PEI was purchased from Polyscience (Warrington, PA) and used to prepare a 1mg/ml stock solution. To dissolve PEI, de-ionized H2O was heated to ∼80° C and mixed with 1mg of PEI. In addition, the solution was cooled down to room temperature, the pH was adjusted to pH 7.2 and the solution was filtered using a 0.22 µm filter (Merck Millipore). Stock solution of linear PEI were stored at -20° C and thawed and stored at 4° C while in use. 2.7. Stability studies Colloidal stability and agglomeration of the polymer solely and siRNA complexed with the polymer were investigated by incubating the polymer in PBS (pH = 7.4) for 4 days and siRNApolymer complexes in PBS containing 10 % fetal calf serum (FCS) for 1 h, 6 h and 24 h at 37 ° C, respectively. At each time point, an aliquot of nanoparticle (NP) solutions was collected to measure NP size. All samples were analyzed by dynamic light scattering based on three independent measurements. 2.8. Cell culture HeLa cells expressing green fluorescent protein (GFP) were purchased from Cell Biolabs (catalog number AKR-213) and adapted to grow in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen catalog number 31870-025). All HeLa cells used in the experiment were cultured in D-MEM containing 10 % FCS, 1 % GlutaMaxTM, 1 % NEAA, 1 % P/S and 1% Blasticidin. HEK cells were cultured in D-MEM containing 10 % FCS, 1 % GlutaMaxTM, 1 % NEAA and 1 % P/S. Cultures were maintained at a temperature of 37° C in a humified 5 % CO2 atmosphere. 2.9. Transfection procedure Green fluorescent protein expressing HeLa cells were transfected in 24-well plates (Corning) using either pentablock based micelleplexes or the positive control LipofectaminTM RNAiMax


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according to the manufacturers protocol. Cy3-fluorescently labeled siRNA and non-labeled siRNA directed against GFP as well as siRNA of random sequence was synthesized by Microsynth (Balgach, Switzerland). The siRNA sequence targeting GFP is 5′-GCA GCA CGA CUU CUU CAA G-3′ (sense) and 5′-CGU CGU GCU GAA GAA GUU C-3′ (antisense). Polymers were dissolved in Ethanol and gently mixed with 100 µl 10 mM PBS pH 7.4 containing 40 pmol siRNA at the desired charge ratio. The mixtures were further incubated for 1 h at room temperature. Polymer siRNA complexes were used immediately after preparation. Complexes were prepared inside the well after which cells (10,000 cells/well) and medium were added. The cells were washed after 24h with PBS and fresh growth medium was added following incubation at 37 °C for additional 48 h. Quantification of downregulation of target gene expression was analyzed by western blot analysis. 2.10. Western blot HeLa cells were washed twice with ice-cold PBS and scraped into 1 ml cold PBS. The cells were pelleted by centrifugation at 600× g for 5 min and resuspended using 100 µl RIPA buffer containing protease inhibitors. The pooled cells were lysed mechanically and rotated for 30 min at 4°C. To remove nuclei, the samples were spun down (20,800× g, 15 min) and the protein concentration of the supernatant was determined by DC protein assay (Bio-Rad). Then, 30 µg of protein/well were loaded on 12% gels and separated by SDS-PAGE. Transfer to nitrocellulose membranes was applied at 100 V for 1.5 h, and the blots were blocked for 1 h with 5% milk powder in TBS-Tween. Primary antibodies were applied overnight, secondary antibodies for 1 h. The immunoblots were detected by enhanced chemiluminescence (Pierce) on Kodak BioMax light films (Sigma). 2.11. Cellular uptake determination of siRNA


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To visualize the cellular uptake of siRNA, the cells were transfected with complexes containing Cy3-labeled siRNA. HeLa and HEK cells were incubated with non-targeted and targeted Cy3-siRNA/polymer, Cy3-siRNA/lipofectamine complexes and naked Cy3-labeled siRNA. Cells were seeded in 24-well plates in medium containing 10 % FCS for 24 h. The naked Cy3-siRNA, Cy3-siRNA/polymer and Cy3-siRNA/lipofectamine complexes were added to each well, and the cells were incubated at 37 °C for 4 h and 24 h, respectively. The culture medium was removed and the cells were washed with PBS (pH = 7.4). The cells were fixed with 4 % Paraformaldehyde (PFA) for 10 min. The cell nuclei were stained with 4',6-diamidino-2phenylindole (DAPI) solution for 20 min at room temperature. Subsequently, the cells were washed with PBS two times and then mounted with fluorescence mounting medium (Dako, Carpinteria, CA). The cells were visualized using a fluorescence microscope (Olympus BX61 Diana) under a magnification of 20. 2.12. Cytotoxicity assay The cytotoxicity of pentablock based micelleplexes was evaluated with HeLa cells by measuring cell viability using the Resazurin reduction assay. Briefly, cells were seeded in 100 µl media in 96-well microtiter plates at a density of 4000 cells /well. Following overnight incubation, the cells were exposed to a range of different concentrations of pentablock based micelleplexes and grown at 37°C under a 5% CO2 atmosphere for 24 h and 48 h. Then, 6 µl of 0.02% (w/v) Resazurin (Sigma-Aldrich, R7017) in phosphate buffered saline (PBS) was added to each well and incubation was continued for an additional 1 h. Finally the fluorescence was read using a spectramax GEMINI XS microplate reader (λexc = 544 nm, λem = 590 nm). 2.13. Statistical analysis Standard statistics includes calculation of means and standard deviations (SD) and Student’s


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t-test for group comparisons. In all experiments, p < 0.05 was considered statistically significant. 3. Results and Discussion 3.1. Design and Synthesis of block copolymers As aforementioned, an optimal nano gene delivery system should take its cytotoxicity and stability into consideration in advance, in respect of which shielded charge and targeted delivery are frequently utilized. Following the criteria, we have arranged a novel nano siRNA delivery system depicted in Figure 1. The system is composed of three components, i.e., penta-block copolymer

PMOXA-PABOXA-PDMS-PABOXA-PMOXA

as

a

matrix

for

siRNA

encapsulation, and tri-block copolymer FA-PMOXA-PDMS-PMOXA-FA for a function of targeting as well as siRNA as a cargo. Hereinto, siRNA can be loaded to penta-block copolymer PMOXA-PABOXA-PDMS-PABOXA-PMOXA based micelles via electrostatic interaction, shielded by outer PMOXA segment as well. Meanwhile, ligand FA can be well installed on the surface of resulting micelles via a hydrophobic interaction from the PDMS segments of two copolymers. As a result, a promising siRNA nano delivery system can be constructed as shown in Figure 1. The following is how to build the two block copolymers and prove the concept. In this study, we have synthesized penta-block copolymer PMOXA-PABOXA-PDMS-PABOXAPMOXA 3 as the matrix for constructing the nanocarriers and folic acid conjugated triblock copolymer PMOXA-PDMS-PMOXA 6 (FA-PMOXA-PDMS-PMOXA-FA) as the ligand conjugate for the specifically cellular recognition. The whole syntheses were described in schemes 1 and 2, respectively. Generally, living cationic polymerization is the main procedure to construct the co-block polymers using intermediate poly(dimethylsiloxane) (PDMS) di-triflate 1 as a macro-initiator which can be provided by directly triflation of starting material HO-PDMSOH. After initiation of the polymerization, chain propagation can start in presence of different


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monomers. As shown in scheme 1, micro-initiator 1 firstly reacted with monomer 2-(4azidobutyl)-oxazole22 to produce new initiator, which further reacted with second monomer 2methyl-oxazole (MOXA). As shown in scheme 2, macrointiator 1 just reacted with 2-methyloxazole. Terminating the polymerization is the last step for the synthesis of copolymer. Pentablock copolymer 2 containing pendent azido group and hydroxyl terminal group was provided by quenching the polymerization with aqueous triethylamine solution in high yield. In scheme 2, triblock copolymer PMOXA-PDMS-PMOXA 4 containing azido terminal group was formed in high yield using sodium azide as a quenching reagent. Since the important intermediates 2 and 4 are available now, the final compounds 3 and 6 can be obtained via correspondingly functional transformation and related conjugation-reaction. For the reduction of azide to amine, several methods have been tired, such as Staudiger reaction, palladium catalysed hydrogenation. Due to the difficulty of removing side products (Staugider reaction) and the destruction of PDMS segment (Hydrogenation), a mild reduction condition has to be explored. Fortunately, we found the azido function group from copolymer 2 can be readily reduced to primary amino group by 1, 3-propanedithiol with a simple procedure. Then copolymer 3 was produced in an excellent yield (scheme 1). “Click reaction” between an azide group and alkyne group is very powerful method to conjugate functional ligands to active molecules, such as polymers, proteins and DNA etc.23 As described in scheme 2, folic acid conjugated PMOXA-PDMS-PMOXA 6 (FA-PMOXAPDMS-PMOXA-FA) was synthesized by “click reaction” between the copolymer 4 and an important intermediate-propargylamine modified folic acid 5 using copper sulphate as a catalyst. The final copolymers 3 and 6 are well characterized by 1HNMR and FT-IR. According to the 1

HNMR spectrum of the two copolymers, the peak at around 0 ppm belongs to the signal of

PDMS segment and peaks at around 2.1 ppm and 3.5ppm belong to the signals of PMOXA. The


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signals from functional groups also can be well distinguished, such as, primary amine (~2.65ppm) and folic acid residue (in the experimental part and Figure S1). In addition, the infrared (IR) spectrum also proved the existence of azide functional group for intermediates 2 (~2097 CM-1) and 5 (characteristic peak: ~2104CM-1). On the other hand, the molecular weights of all polymers were deduced from the relevant 1HNMR analysis. As shown in the experimental part and Figure S1, the methylene group (-OSi(Me)2-CH2-CH2-) for each copolymer can be recognized by the signal around 0.55ppm. According to the integration of this signal as a standard, the molecular formulas and these corresponding molecular weights can be deduced and calculated, respectively.

O

HO

Si

O

Si

Tf2O TfO OH 2, 6-lutidine

O 73

O

Si

O

Si

O

OTf

73

HO-PDMS-OH

1

N O

1. O

N3 ; 2.

O

N;

O N

HO

3. H2O/Et3N

O

N 10

O

Si

O

Si

O 73

3

O

O

O Et3N/MeOH

OH 10

N3

SH

HO

3

2

N3 HS

N

N

N

O

N 10

O

Si

O

Si

O O

73

3

NH2 or (NH3)

N

N 3

OH 10

NH2 or (NH3)

PMOXA-PABOXA-PDMS-PABOXA-PMOXA 3

Scheme 1. Synthesis of penta-block copolymer PMOXA-PABOXA-PDMS-PABOXA-PMOXA 3

containing

pendent

primary

amino


function

group.

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O

HO

Si

O

Si

Tf2O OH TfO 2, 6-lutidine

O 73

O

Si

2. NaN3

O

N

Si

O

Si

20

O

O

N

73

O

HN N N N

CO2H

O

N O

CuSO4 . 5H2O

Si

O

Si

O

N

73

20

O

N N N 20

HN O HO HN

N N N

NH O

CO2H HN

N NH2

OTf

NH2 N N N HO2C O HO N HN N N3 H O N 5 20 H

PMOXA-PDMS-PMOXA 4

O

O

2

N N3

Si 73

1 1. O

O

O HO N

Folic acid-conjugated PMOXA-PDMS-PMOXA 6 (FA-PMOXA-PDMS-PMOXA-FA)

NH2

N

N N

HN

Scheme 2. Synthesis of folic acid conjugated tri-block copolymer PMOXA-PDMS-PMOXA 6 (FA-PMOXA-PDMS-PMOXA-FA). 3.2. Preparation and characterization of nanoparticles The pentablock based micelleplexes were prepared upon self-assembling in aqueous solution at the desired N/P ratio as shown in Figure 1. Dynamic light scattering (DLS) data showed a hydrodynamic diameter of 21 ± 3 nm (Figure 2). The slightly smaller hydrodynamic diameter (18 ± 5 nm) observed by TEM analysis can be explained by the fact that the samples are dried during sample preparation, which leads to a decrease in particle size. To examine the loading capacity of the pentablock based micelleplexes, a gel retardation assay was employed (Figure 2). The quantity of siRNA was set as a fixed value, whereas the polymer concentration was increased accordingly to the N/P ratio. Complexation of siRNA with FAPMOXA-PDMS/PMOXA-PABOXA-PDMS based micelles completely prevented siRNA migration at an N/P of 5, indicating full neutralization of the negative siRNA charge. Fully


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complexed siRNA stayed in the gel loading wells due to their large size of the complexes. In addition, the influence of the FA-PMOXA-PDMS polymer on the complexation with siRNA was tested (Figure S3). The data revealed, that FA-PMOXA-PDMS itself has no influence on the complexation with siRNA. The zeta potential of the polyplexes was determined by electrophoretic mobility and reveals that the particle surface of the polyplexes is slightly positive (+ 4 mV) before addition of siRNA, indicating that most of the positive charge is shielded by the outer PMOXA shell. It has been proven that nanoparticles with almost neutral surface charge (zeta potential between – 10 and + 10 mV) are less likely to affect immunological reaction in vivo.24,

25

As a result, the

micelleplexes, prepared at N/P 5, with a moderate positive charge (3 ± 1 mV) and small size (21 ± 3 nm) were selected for further biological experiments.

Figure 1. Illustrative representation of siRNA pentablock copolymer formation.


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Figure 2. Characterization of the size and siRNA loading capacity of the micelleplexes. (A) Size determination by TEM. (B) Polymer based complexes at different N/P ratios were loaded onto agarose gel and electrophoresis was carried out. (C) Hydrodynamic size of micelleplexes measured by DLS. The FA-PMOXA-PDMS/PMOXA-PABOXA-PDMS micelleplexes have a diameter of 21 ± 3 nm (DLS) or 18 ± 5nm (TEM). 3.3. Stability studies Cellular interactions of nanoparticles are well known to be based on physicochemical properties such as size, surface charge, structure and chemical composition of the particles.26-28 One of the key factors, which directly relates to cellular uptake is the surface charge of NPs.29 Positive charged NPs are strongly being taken up by the cells due to unspecific electrostatic interactions with negatively charged sugar moieties of monosaccharides and polysaccharides on the cell surface. Furthermore, the use of positive charged NPs in biological systems is often constrained due to the inherent toxicity associated with them.30 In regard to in vivo administration of the NPs, the positive surface charge often lead to binding with biomolecules


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such as serum proteins in biological fluids.31 Cationic polymers are well known to form aggregates in serum containing medium, which turns them highly unfavorable for further in vivo applications. Therefore, it is important while designing new vectors for siRNA delivery to consider the physicochemical properties to construct optimal nanoformulations for efficient in vitro and in vivo use. To evaluate the suitability of the micelleplexes for potential biomedical applications, the studies of stability regarding long-term storage, degradation and serum interactions were carried out. To evaluate the long-term stability, the NPs were prepared in PBS (pH = 7.4) at 37 ° C for 4 days and the size and zeta potential were measured as shown in Figure 3. During the study there was no significant change in the average diameter of the NPs whereas the surface charge decreased within time, which might be explained by the increased absorption of negative ions from the buffer solution such as phosphate ions. The stability in biologic fluids was assessed by means of aggregation testing of the pentablock based micelleplexes and the polymer/siRNA complexes in 10% serum, and was measured by DLS measurements. For the detection of nanoparticle aggregation, we chose to use DLS intensity distribution instead of the more popular number distribution, which is usually being used for nanoparticle characterization, due to the high sensitivity of the detection of larger particles such as aggregates compared to single particles. Based on the Rayleigh scattering approximation, the intensity of the scattered light by a particle is proportional to the sixth power of its diameter. Therefore, larger particles such as aggregates will be easily detected by the intensity distribution even though the total numbers within the whole sample is small. As seen in Figure 4A, measurement of 100% serum shows two peaks at ~10 and 50 nm, which are usually observed due to the high concentration of proteins in the serum as described by van Gaal et. al.32


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Pentablock based micelleplexes and 25 kDa Polyethylenimine (PEI) were tested in serum containing buffer solution and analyzed after 1 h, 6 h and 24 h. As seen in Figure 4B, after addition of the micelleplexes to the serum containing buffer solution, no significant change of the intensity spectra for large aggregates could be detected after all measured time-points. The micelleplexes are similar to the size of serum proteins but have in comparison of much lower concentrations. On the contrary, the highly cationic polymer PEI, which is one of the most successful and widely used polymers for gene delivery, shows an additional peak at much larger size, indicating aggregate formation. This observation could be assured by TEM images showing aggregate formation as compared to the pentablock based copolymers (Figure 4D - E). The high colloidal stability of the micelleplexes can be attributed to their architecture. The PMOXA outer shell masks most of the positive charge of the PABOXA block, therefore reducing unspecific interactions with serum proteins. Former studies have evaluated that nanoparticles with zeta potential in the range between - 10 and + 10 mV are less likely to cause immunological reactions.24, 25 Furthermore, after loading of siRNA onto the micelleplexes, a decrease of the zeta potential can be observed compared to the naked pentablock copolymer micelleplexes turning them slightly negative (- 1 mV). These studies demonstrated the excellent stability of pentablock based micelleplexes for potential in vivo application.


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Figure 3. Long-term stability conformation of micelleplexes. Stability conformation over 4 days indicating no significant change in the average diameter (blue), whereas a decrease of the zeta potential (red) can be measured. The data shown are the mean and SD from three different experiments (n = 3)


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Figure 4. Characterization of the colloidal stability of the micelleplexes. (A) Hydrodynamic size measurements after 24 h of serum, Polymer + 10% serum, siRNA/Polymer + 10% serum and 25 kDa PEI in 10% serum. As compared with the micelleplexes, the hydrodynamic size pattern of the highly cationic PEI shows an aggregate peak. Hydrodynamic size measurements of micelleplexes (B) and 25 kDa PEI (C) at 1 h, 6 h and 24 h in 10% serum. TEM images of 10% serum (D) micelleplexes (E) and 25 kDa PEI (F) after 24 h in buffer, containing 10% serum, demonstrating aggregate formation of PEI. The data shown (A – C) are the mean from three different experiments (n = 3). 3.4. Cellular uptake of siRNA We compared the cellular uptake efficiency of non-targeted and targeted siRNA/pentablock copolymer complexes on cancerous folate receptor (FR) positive (HeLa) or normal FR-negative HEK293 cells. The cellular uptake of siRNA was observed with Cy3 fluorescently labeled siRNA. Naked Cy3-siRNA was employed as negative control for uptake study. HeLa and HEK cells were incubated with naked Cy3-siRNA, non-targeted and targeted Cy3-siRNA/polymer as well as Lipofectamin/Cy3-siRNA complexes for 4 h and 24 h at 37 °C. As compared to the nontargeted complexes, the red fluorescent Cy3-labeled siRNA complexes were strongly observed in HeLa cells treated with FA targeted complexes after 24 h (Figure 5A). These results indicate that the cellular uptake of the FA targeted complexes is mainly based on a folate-receptor-mediated endocytosis mechanism, but further of FA specific targeting competitive studies using free FA will need to be considered for further studies. A small amount of Cy3-siRNA can be detected in HeLa cells treated with non-targeted complexes, which might be explained by fluid phase endocytosis, whereas particles are spontaneously being taken up through invagination of the cell membrane.33


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The normal FR-negative HEK293 cells showed no significant signal regarding cellular uptake of the Cy3-siRNA, for the targeted and the non-targeted complexes even after 24 h (Figure 5B) as expected, due to the lack of the folic acid receptor expression as proven by western blot analysis (Figure 6B). However, the proper mechanism of cellular uptake still remains a topic of research.


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Figure 5. Cellular uptake study of siRNA. Fluorescence microscopy images showing siRNA uptake of FR-positive HeLa (A) and of normal FR-negative HEK293 (B) cells, after 4 h and 24 h, respectively. DAPI staining, shown in blue, was added to visualize nuclei. Red signals represent Cy3-labeled siRNA molecules. Note that Lipofectamin binds unspecifically to cells, one feature precluding its use in targeted “in vivo” contexts. 3.5. Gene silencing mediated by siRNA loaded pentablock copolymer nanoparticles. To evaluate the gene knockdown efficiency of the polymer complexes, we used stably green fluorescent protein (GFP) expressing HeLa cells and siRNA targeting GFP as a model. Hela cells highly overexpress folic acid receptors as compared to HEK cells (Figure 6B). Visualization of the gene silencing was done by western blot analysis and fluorescence microscopy. GFP expression reduction was analysed by normalizing with tubulin expression. GFP expression was suppressed to 92 ± 4.2% and 69 ± 5.5% using non-targeted PMOXA-PABOBA-PDMS polymer and FA-PMOXA-PDMS/PMOXA-PABOBA-PDMS polymer, respectively. In comparison, the


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common transfection reagent Lipofectamine showed strong protein suppression to 46 ± 7% (Figure 6B). Adsorption of serum proteins on the nanoparticle surface may hinder ligand– receptor interaction and therefore preventing cellular uptake.34, 35 However, these results demonstrate that introduction of a ligand clearly enhanced siRNA delivery and knockdown. Even though the decrease is not as high as seen in Lipofectamine, Lipofectamine itself undergoes a non-specific cellular uptake route and is therefore not suitable for in vivo administration application when receptor targeted delivery strategies are pursued. The gene knockdown efficiency can be qualitatively directly correlated with the uptake efficiency. Whereas lipofectamine shows a high cellular uptake and high level of GFP expression suppression, the targeted and non-targeted polymer/siRNA complexes lead to less cellular uptake and lower levels of protein expression suppression. Furthermore, no silencing effect was confirmed in all complexes using non-targeted siRNA (Figure S4). Our results show also that siRNAs were localized to perinuclear regions and this localization was correlated to the silencing efficiency (Figure 6C). Detection of GFP (TRITC) by fluorescence microscopy revealed that GFP is translocating to the nucleus. The translocation of GFP into the nucleus, due to multimeric variance of GFP found in GFP overexpressing cell lines, has been described by Seibel et al.36 In addition, a slight difference in the fluorescence pattern of the taken up siRNA by Lipofectamine or the micelleplexes can be seen. The micelleplexes show a more even distribution of the siRNA within the cytoplasm, whereas the Lipofectamine samples show concentrated dot like structures, localized around the periphery of the nucleus. This pattern might be explained by the fact that non-targeted micelleplexes are also been taken up by the cells, possibly by fluid phase endocytosis, whereas their intracellular fate might differ from the one of


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Lipofectamine. These observations possibly correlate, that RNAi activity is associated with siRNA localization to specific compartments in the cytoplasm, such as the perinuclear localization, as also hypothesised by others.37

Figure 6. Gene silencing evaluation of siRNA (red), targeting GFP (green) using non-targeted (PMOXA-PABOXA-PDMS) and targeted siRNA/polymer (FA-PMOXA-PDMS/PMOXAPABOXA-PDMS) complexes compared with the classic transfection agent Lipofectamine. (B) Western blot showing protein expression of FR in HeLa and HEK293 cells as well as GFP


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knockdown in HeLa cells. 30 µg of protein from total lysates were loaded and subjected to WB analysis. (C) Fluorescent microscopy images showing cellular uptake of the Cy3-siRNA (TRITC) and the decrease in GFP expression (TRITC). Data were normalized versus an untreated control. The results are representative of three independent experiments (n = 3). Statistical significant difference to negative control (cells) is denoted by asterisks, where ** p < 0.01, and *** is p < 0.001. 3.6. In vitro cytotoxicity evaluation Toxicity is an important consideration in the design of nanoparticles for efficient nucleic acid delivery. The toxic effect in nucleic acid delivery by polymers is mostly based on the cationic nature of the carrier system and often combined with high transfection efficiency.38 Therefore, many approaches have been conducted using polyethylenglycol (PEG) to prevent cationic nanoparticles from unwanted interactions and lower cytotoxicity. However, although PEGylation of NPs has been claimed to be safe in particular, recent findings have shown that PEG might lead to complement activation related pseudoallergic reactions and should be considered for design of new delivery systems.7 Cytotoxicity of the pentablock based polyplexes was assessed with HeLa cells by measuring cell viability using the Resazurin reduction assay. As shown in Figure 7 cytotoxicity detection was at much elevated polymer concentrations than the one used in the cell studies (5 µM). Cell viability was greater than 80% at polymer concentration less than 100µM. The low level of cytotoxicity can be explained by the architecture of the pentablock copolymer. The positively charged polymer PABOXA block is surrounded by a hydrophilic PMOXA polymer layer, which generates a shielding effect of the nanoparticle from direct charge induced interactions with the cells. Even though under the shielding effect, the polymer based polyplexes seemed to cause higher toxicities at very higher concentrations (e.g., 1000µM and 5000 µM).


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Figure 7. (A) Viability assessment of HeLa cells treated with micelleplexes at various concentrations for 24 h (blue) and 48 h (red). The cell viability is still above 90 % after 48 h at concentrations 10 x higher than the one used in the experiments compared to untreated cells. (B D) Light microscopy images of HeLa cells treated with different polymer concentrations. 4. Conclusion In summary, we have designed and synthesized a new type of amphiphilic copolymers PMOXAPABOXA-PDMS-PABOXA-PMOXA 3 and its precursor 2, both of which own highly reactive function groups, i. e. amine and azide, respectively. One application was applied by using the positively charged copolymer 3 for gene encapsulation. As a result, the pentablock-copolymer based micelleplexes of FA-PMOXA-PDMS/PMOXA-PABOXA-PDMS for efficient targeted siRNA delivery (in vitro) has been developed. The currently achieved 31 % knockdown 27 Environment ACS Paragon Plus

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efficiency shows its possible potential regarding gene therapy. However, quantification of the mRNA knockdown needs additional methods such real time quantitative RT-qPCR to allow the extent of the knockdown to be quantified.39 The pentablock architecture allows the formation of highly stable micelleplexes with a neutral surface charge, siRNA condensation properties, excellent colloidal stability in serum and good in vitro biocompatibility, due to the absence of considerable cytotoxicity. Furthermore, selective delivery of the siRNA could be proven by the introduction of a ligand linked block copolymer. Since cell lines show different cellular uptake mechanisms, competitive studies using free FA need to be conducted for further proof of FA specific targeting. However, this pentablock based system design for siRNA delivery might provide a great feasible and potential platform to be applied in vivo for cancer gene therapy. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 1

HNMRs of compounds 2, 3, 4, 6; 1HNMR studies of copolymer 4 in D2O and CD3OD;

Determination of FA-Polymer interactions on the loading capacity of the micelleplexes; Gene silencing effect using non-targeted siRNA. AUTHOR INFORMATION Corresponding Author * E-mail address: [email protected]; Tel: +41 61 26 55 581 or +41 61 26 52 525. Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is financially supported by the Swiss National Science Foundation research program NRP 62 “Smart Materials”. The authors thank V. Oliveri and C. Alampi of the Zentrum für Mikroskopie Basel (ZMB) for TEM-measurements and the lab of Prof. M. Wymann from the Department of Biomedicine for providing the HEK293 cells. ABBREVIATIONS siRNA, small interfering RNA; PMOXA, poly-2-methyl-oxazole; PABOXA, aminobutyl)-oxazole; PDMS, poly(dimethylsiloxane); PMOXA,

poly-2-(4-

FA, Folic acid; PMOXA-PDMS-

poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyl-oxazoline);

PMOXA-PABOXA-PDMS-PABOXA-PMOXA,

poly(2-methyloxazoline)-b-poly-2-(4-

aminobutyl)-oxazoleb-b-poly(dimethylsiloxane)-b-poly-2-(4-aminobutyl)-oxazole-b-poly(2methyl-oxazoline); FA-PMOXA-PDMS-PMOXA-FA, folic acid-poly(2-methyloxazoline)-bpoly(dimethylsiloxane)-b-poly(2-methyl-oxazoline)-folic acid;

PEI, Polyethylenimine ; GFP,

Green Fluorescent Protein; PEG, polyethylenglycol. REFERENCES (1)

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(2)

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Miele, E.; Spinelli, G. P.; Miele, E.; Di Fabrizio, E.; Ferretti, E.; Tomao, S.; Gulino, A.

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Efficient Receptor Mediated siRNA Delivery in Vitro ... - ACS Publications

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