European Journal of Pharmaceutical Sciences 75 (2015) 60–71

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system C. Sardo a, R. Farra b, M. Licciardi a, B. Dapas c, C. Scialabba a, G. Giammona a, M. Grassi b, G. Grassi c,⇑, G. Cavallaro a a b c

Dipartimento di Scienze e Tecnologie Biologiche, Chimiche, Farmaceutiche (STEBICEF), Lab of Biocompatible Polymers, University of Palermo, via Archirafi 32, 90123 Palermo, Italy Department of Engineering and Architecture, University of Trieste, Via Alfonso Valerio, 6/A, I-34127 Trieste, Italy Department of Life Sciences, University Hospital of Cattinara, Strada di Fiume 447, 34100 Trieste, Italy

a r t i c l e

i n f o

Article history: Received 2 December 2014 Received in revised form 28 February 2015 Accepted 24 March 2015 Available online 4 April 2015 Keywords: Inulin Diethylenetriamine (DETA) Inu-DETA copolymer siRNA JHH6 16HBE

a b s t r a c t Small interfering RNAs (siRNAs) have the potential to be of therapeutic value for many human diseases. So far, however, a serious obstacle to their therapeutic use is represented by the absence of appropriate delivery systems able to protect them from degradation and to allow an efficient cellular uptake.In this work we developed a siRNA delivery system based on inulin (Inu), an abundant and natural polysaccharide. Inu was functionalized via the conjugation with diethylenetriamine (DETA) residues to form the complex Inu-DETA. We studied the size, surface charge and the shape of the Inu-DETA/siRNA complexes; additionally, the cytotoxicity, the silencing efficacy and the cell uptake-mechanisms were studied in the human bronchial epithelial cells (16HBE) and in the hepatocellular carcinoma derived cells (JHH6).The results presented here indicate that Inu-DETA copolymers can effectively bind siRNAs, are highly cytocompatible and, in JHH6, can effectively deliver functional siRNAs. Optimal delivery is observed using a weight ratio Inu-DETA/siRNA of 4 that corresponds to polyplexes with an average size of 600 nm and a slightly negative surface charge. Moreover, the uptake and trafficking mechanisms, mainly based on micropinocytosis and clatrin mediated endocytosis, allow the homogeneous diffusion of siRNA within the cytoplasm of JHH6. Notably, in 16 HBE where the trafficking mechanism (caveolae mediated endocytosis) does not allow an even distribution of siRNA within the cell cytoplasm, no significant siRNA activity is observed. In conclusion, we developed a novel inulin-based siRNA delivery system able to efficiently release siRNA in JHH6 with negligible cytotoxicity thus opening the way for further testing in more complex in vivo models. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Short interfering RNAs (siRNAs) are double stranded RNA molecules (19–27 bp) that can bind via their antisense strand (Grassi et al., 2013, 2010, Scaggiante et al., 2011; Agostini et al., 2006) a complementary region of a cellular mRNA inducing mRNA degradation. Compared to the conventional therapeutic drugs, siRNAs represent an emerging paradigm for the treatment of many human diseases such as cancer (Farra et al., 2011, 2010; Resnier et al., 2013), cardiovascular, neurodegenerative, metabolic diseases and viral illnesses (Werth et al., 2010; Dapas et al., 2009; Racchi et al., 2009; Deng et al., 2014). However, owing to their negative

⇑ Corresponding author at: Department of Life Sciences, University Hospital of Cattinara, Strada di Fiume 447, 34100 Trieste, Italy. Tel.: +39 040 3996227; fax: +39 040 3994593. E-mail address: [email protected] (G. Grassi). http://dx.doi.org/10.1016/j.ejps.2015.03.021 0928-0987/Ó 2015 Elsevier B.V. All rights reserved.

charges, siRNA molecules are not readily taken up by cells that have a negatively charged surface. Moreover, because of their hydrophilic nature, siRNAs poorly diffuse across the phospholipidic bilayer of cell membrane. Finally, siRNAs are susceptible to nuclease degradation, a fact that limits the effective administration through most of the routes (Li et al., 2006; Zhang et al., 2007; Deng et al., 2014). Once inside the cells, a significant fraction of siRNAs tends to be sequestered into endosomes thus reducing the amount of free active molecules. For these reasons, much effort has been put in the development of safe and efficient siRNA delivery systems. Nano-devices effectively enhance the bioavailability of loaded siRNAs improving their permeability through bio-membrane and minimizing the negative effects of the biologic environment. Among the materials used for nano-devices-mediated delivery of siRNAs, cationic polymers emerged as one of the most versatile and effective option (Ballarin-Gonzalez and Howard, 2012). Cationic polymers can form with siRNAs polyplexes,

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colloidal systems originated from the electrostatic interactions between the negative nucleic acids and the positive charged polymers (Cavallaro et al., 2014; Sardo et al., 2014). Once in the polyplexes, siRNA resistance to nuclease degradation, cellular uptake and endosome escape (Kaneda, 2001; Brown et al., 2001) are significantly improved. In our previous study (Sardo et al., 2014), due to the considerable interest in the use of renewable resources for biomedical applications, we synthesized a sperminated polycation using the Enhanced Microwave Synthesis (EMS) (Sardo et al., 2014). This last is a novel synthesis approach more efficient than the conventional thermal heating. As backbone, we employed inulin, a natural polysaccharide considered abundant, biocompatible and susceptible to chemical modifications for the specific requirements of siRNA delivery. The encouraging results obtained in term of siRNA delivery, prompted us to explore the possibility to develop a novel oligoamine-modified inulin delivery system for siRNAs. The novel polycation consists of an Inulin-backbone conjugated with Diethylenetriamine residues (Inu-DETA). In this work, we evaluated the interaction of Inu-DETA with siRNAs as well as the size, surface charge and shape of the polymer/siRNA complexes. Additionally, we studied the cytotoxicity, transfection efficacy/cell uptake-mechanism and the silencing capacity of the Inu-DETA/ siRNAs complexes. Collectively, our data indicate that the novel Inu-DETA polymer is a valuable tool for siRNA delivery. 2. Materials and methods 2.1. Materials Inulin from Dahlia Tubers Mw  5000 Da and DETA were purchased from Fluka. Anhydrous N,N-dimethylformamide (a-DMF) was from VWR; Bis(4-nitrophenyl) carbonate (4-BNPC) and agarose were purchased from Sigma-Aldrich. 1H NMR spectra were recorded in D2O (VWR) using a Bruker AC-250 spectrometer operating at 250.13 MHz. 2.2. Cell culture 16HBE human bronchial epithelial cells or JHH6 hepatocellular carcinoma cells (Baiz et al., 2014) were grown in Dulbecco’s modified Eagle’s medium (DMEM) and William’s medium, respectively, with 10% fetal bovine serum (FBS) and 1% of penicillin/streptomycin (100 U/ml penicillin and 100 mg/ml streptomycin), at 37 °C in 5% CO2 humidified atmosphere. DMEM and the other cell culture constituents were purchased from Euroclone.

Ò

filtration chromatography using a mixture 1:1 of Sephadex G-25 Ò and Sephadex G-15 as gel filtration medium and double-distilled water as eluent. After recovering, the solution was freeze-dried. The pure product was characterized by 1H NMR analysis in D2O. 1 H NMR of Inu-DETA (D2O) reveals peaks at d: 2.7 (m, 4HDeta, –CH2–NH–CH2–), 2.8 (m, 2HDeta, –CH2–NH2), 3.2 (m, 2HDeta, –O– CO–NH–CH2–), 3.5–4.0 (m, 5HInu, –CH2–OH; –CH–CH2–OH; –C–CH2–O–), d 4 (t, 1HInu, –CH–OH), d 4.2 (d, 1HInu, –CH–OH). 2.4. Size exclusion chromatography (SEC) characterization The weight-average molecular weights (Mws) and polydispersity (Mw/Mn) of inulin and Inu-DETA, were determined by aqueous SEC. The protocol involved a PolySep-GFC-P3000 gel column from Phenomenex connected to a Waters 2410 refractive-index detector. Phosphate buffer solution 0.05 M at pH 4 was used as the eluent at 35 °C with a flow rate of 0.6 ml/min. Pullulan standards (in the range 180–47,300 Da), used for the calibration procedure and for sample evaluation, were dispersed at a concentration of 2.5 mg/ml in the mobile phase; all samples were filtered through a 0.45 lm cellulose acetate filter prior to analysis. 2.5. Buffering capacity obtained by acid-base titration To determine the relative buffering capacity of Inu-DETA polymer, acid-base titration was performed. The samples were prepared by suspending 6 mg of Inu-DETA copolymer at a concentration of 0.2 mg/ml in double distilled water. The pH was adjusted to nearly 10.0 with 0.1 N sodium hydroxide and then the solution was titrated by adding gradually 30 ll of 0.1 N HC1 until reaching a pH of 3. Titrations of unmodified inulin (0.2 mg/ ml), and DETA, at the same concentration present in Inu-DETA copolymer, were also evaluated. The slope of the curve obtained plotting the pH values versus the amount of HCl consumed, provided an indication of the buffering capability of the copolymer Inu-DETA. The relative Buffering Capacity (RBC7.4–5.1), defined as the percentage of amine groups becoming protonated within the pHrange from 7.4 to 5.1, was calculated according to the following equation:

RBC7:45:1 ¼ ðV HCl  0:1MÞ  100=N mol where VHCl is the volume of 0.1 M HCl employed to change the pH values of the polymer solutions from 7.4 to 5.1 and Nmol is the total moles of protonable amine groups in the analyzed Inu-DETA copolymer solution. 2.6. Gel retardation assay

2.3. General procedure for the synthesis and characterization of InulinDiethylenetriamine copolymer via EMS Inulin was dried in an oven at 70 °C for 24 h. Thereafter, 250 mg of dried inulin (corresponding to 1.5 mmol of fructose repeating units) were dissolved in 4 ml of a-DMF and, after the addition of 234 mg of 4-BNPC (previously dissolved in 1 ml of the same solvent), the mixture was placed in a CEM Discover Microwave Reactor. After 1 h irradiation at 25 W and at 60 °C, the reaction mixture was added slowly and drop wise, to a-DMF DETA solution (381 ll DETA in 1 ml a-DMF) using five folds excess more DETA compared to the mmoles of 4-BNPC. Subsequently, the reaction mixture was kept for 4 h at room temperature under constant magnetic stirring; after that, precipitation in a mixture of ethyl ether/dichloromethane 1:1 (v/v) was performed. The suspension was collected by centrifugation and the residue washed twice with 50 ml of the same mixture of solvents. The obtained solid was carefully dried, solubilized in 2 ml of distilled water and purified by gel

Inu-DETA/siRNA polyplexes were formed in nuclease free HEPES buffer pH 7.4, containing 5% glucose (HBG). In particular, 10 ll of Inu-DETA solutions at various concentrations were added to 10 ll of a siRNA solution (0.2 mg/ml) in order to obtain the desired weight ratios. The mixture was mixed by gently pipetting for 10 times, followed by 30 min incubation at room temperature. Fifteen ll of each sample were then loaded on a 1.5% agarose gel, containing 0.5 lg/ml ethidium bromide and run at 100 V in tris-acetate/EDTA (TAE) buffer for 20 min. The gels were then visualized against an UV transilluminator and photographed using a digital camera. 2.7. Measurements of size and Zeta potential for Inu-DETA/siRNA complexes Dynamic light scattering studies (DLS) were performed at 25 °C with a Malvern Zetasizer NanoZS instrument fitted with a 532 nm

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laser at a fixed scattering angle of 173°, using the Dispersion Technology Software 7.02. The Inu-DETA/siRNA polyplexes were prepared in Dulbecco’s Modified Phosphate Buffered Saline (DPBS) nuclease free (pH 7.4), by adding to 50 ll of siRNA solution (0.08 lg/ll), the same volume of Inu-DETA solutions at various concentrations in order to obtain weight ratios in the range of 3– 50. The intensity-average hydrodynamic diameter (nm), and polydispersity index (PI) were obtained by cumulative analysis of the correlation function. Polyplex containing solutions were then diluted with 400 ll of DPBS nuclease free and used to determine the Zeta potential of Inu-DETA/siRNA complexes at the above mentioned weight ratio ranges. Zeta potential measurements were performed by aqueous electrophoresis measurements, recorded at 25 °C using the same apparatus. The zeta potential values (mV) were calculated from the electrophoretic mobility using the Smoluchowsky relationship.

2.8. Morphology of Inu-DETA/siRNA complexes: transmission electron microscopy Samples for Transmission Electron Microscopy (TEM) observations were prepared putting 50 ll of 0.25% (w/v) sample solutions containing Inu-DETA/siRNA polyplexes at a weight ratio of 20, on copper grids. Following the removal of the liquid excess by a filter paper, the grids were allowed to dry spontaneously. The samples were observed using a JEM-2100LaB6 transmission electron microscope operating at an accelerating voltage of 200 kV, equipped with a MultiScanCCDcamera.

2.11. In vitro transfection and determination of target mRNA/protein level The sequences of the siRNAs directed against the transcription factor E2F1 mRNA (siE2F1) and the luciferase mRNA (siGL2, control), were purchased from Eurofins Genomics GmBH, Ebersberg, D. The sequences of these chemically un-modified siRNAs, have been previously reported (Cavallaro et al., 2014; Dapas et al., 2009). The day before transfection, JHH6 cells were seeded at a density of 3.8  103 cells/cm2 in 6 well microplate in the presence of 3 ml of 10% fetal calf serum-containing medium. Optimal transfection conditions were obtained by using an Inu-DETA polymer/ siRNA weight ratio of 4:1 (R4). The mixture polymer/siRNA (200 nM) was then administered to the cells for 4 h at 37 °C in the presence of serum-free medium. Afterward, the transfection medium was removed, cells were washed with 3 ml of PBS and then 4 ml of complete medium were added. The effects on the cell number and on the expression level of the target mRNA were evaluated 3 days thereafter, being this the optimal time point for evaluating siRNA effect (Farra et al., 2011). After this time period, total RNA was extracted, quantified and the quality evaluated as described (Baiz et al., 2014). Reverse transcription was performed using 500 ng of total RNA in the presence of random hexamers and MuLV reverse transcriptase (Applera Corporation, USA). The primers (Eurofins Genomics, 300 nM) and the Real-Time amplification conditions were as described (Baiz et al., 2014). The relative amounts of the E2F1, E2F3, cyclin D1 and cyclin A2 mRNA were normalized by 28S rRNA content. E2F1 protein levels were evaluated by western blotting as described (Farra et al., 2011). 2.12. Uptake studies

2.9. Inu-DETA/siRNA stability in the presence of albumin The stability of Inu-DETA/siRNA polyplexes was determined after polyplexes incubation with albumin. After Inu-DETA and siRNA polyplexes formation in HBG at pH 7.4 for 30 min, the resulting polyplexes (20 ll), were mixed with albumin solution (200 lg/ll in HBG – 5 ll) and incubated at room temperature for 4 or 8 h. Gel electrophoresis was then performed as described in Section 2.6.

2.10. Cytotoxicity studies The Inu-DETA polymer was incubated with cells using six different concentrations (25, 50, 100, 250, 500, 1000 lg/ml). The Inu-DETA/siRNA polyplexes were prepared in sterile nuclease free DPBS at weight ratios of 7, 10, 20, 30 and 40 by mixing equal volumes of Inu-DETA and siRNA solutions. For the Inu-DETA/siRNA polyplexes, after 30 min of complexation, samples were diluted in a final volume of 200 ll of OPTI-MEM and added to 16HBE or JHH6 cells (final siRNA concentration of 200 nM), seeded the day before in a 96 well plate at a density of 1.5  104 cells/well. Following 4, 24 and 48 h of incubation, cells were washed with 100 ll of sterile DPBS and incubated with 100 ll of fresh DMEM containing 20 v% of MTS reagent solution (3-(4,5dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium). Plates were incubated at 37 °C for 2.5 h and then a UV plate reader (Microplate spectrophotometer Multiskan EX – Thermo Scientific) was used to measure formazane absorbance at 490 nm. Untreated cells were used as negative control (100% viability). Wells filled with MTS reagents and DMEM or William’s medium without cells, were used as blank to calibrate the spectrophotometer to zero absorbance. The cell viability was calculated by the formula (Abs sample/Abs control)  100 as % of control.

To determine from the qualitative point of view the cellular uptake of Inu-DETA/siRNA polyplexes, 16HBE or JHH6 cells were seeded in a 48 well plate at a density of 6  104 cells/well and grown as above reported. Twenty-four h later, the culture medium was replaced by 600 ll of fresh OPTI-MEM I medium containing Inu-DETA/siRNA-Cy5 polyplexes at different weight ratios, to reach a final siRNA concentration of 200 nM. After 4 h incubation, cells were extensively washed with sterile DPBS and fixed in 4% paraformaldehyde in DPBS for 30 min. For the quantitative determination of the cellular uptake of InuDETA/siRNA polyplexes, 16HBE or JHH6 cells were grown in a 24 well plate at a density of 1.2  105 cells/well for twenty-four h. After this time the culture medium was replaced with 1.2 ml of OPTI-MEM I medium containing Inu-DETA/siRNA-Cy5 polyplexes at weight ratios of 2, 4 and 7 to reach a final siRNA concentration of 200 nM. After 4 h incubation, cells were extensively washed with sterile DPBS and lysed in 300 ll lysis buffer (2% SDS, 1% Triton X-100, in DPBS) on ice for 30 min. The lysates were divided in two parts: the first one (275 ll) was used to measure the fluorescence intensity by a Shimadzu RF-5301 PC spectrofluorophotometer, the second one (25 ll) to evaluate the total protein amount by BCA protein assay. The results were expressed as the ratio fluorescence intensity (U.A.)/mg protein. 2.13. Effect of cellular uptake inhibitors on Inu-DETA polyplexes cell internalization To optimize the conditions of endocytosis pathway inhibition, we first assessed the cytotoxic effects of the cellular uptake inhibitors. 16HBE and JHH6 cells were seeded in a 96 well plate as reported in 2.10. Twenty-four h later, the culture medium was replaced with 200 ll fresh complete DMEM or William’s medium containing either 1-3 mg/ml methyl-b-cyclodextrin (MbC), or

C. Sardo et al. / European Journal of Pharmaceutical Sciences 75 (2015) 60–71

0.01–1 lM wortmannin (Wo) or 0.1-200 lM phenylarsine oxide (PAO). After incubation times of 0.5, 1 and 4 h, cells were extensively washed with sterile DPBS and treated with MTS containing medium to assess cell viability as described in 2.10. Following dose and incubation time optimization of each endocytosis inhibitor, 16HBE or JHH6 cells were seeded in a 48 or 24 well plate at a density of 6  104 or 1.2  105 cells/well, respectively, and grown as reported in 2.10. Prior to the addition of Inu-DETA/ Cy5-siRNA polyplexes at weight ratios of 2, 4 and 7, cells were pre-incubated at 37 °C with complete culture medium containing 2 mg/ml MbC, 1 lM Wo or 1 lM PAO; the incubation time was of 30 min and 1 h for MbC/Wo and PAO, respectively. Afterward, the medium was replaced with 600 or 1200 ll OPTI-MEM I containing Inu-DETA/Cy5-siRNA polyplexes and the inhibitors, except PAO, at the same concentrations. After 4 h of incubation, cells grown in 48 well plates were fixed for fluorescence microscopy analysis (as reported in Section 2.12), while cells cultured in 24 well plates were lysed to determine lysate fluorescence (as reported in Section 2.12). Non-treated cells were used to set the background fluorescence; cell treated with Inu-DETA/Cy5-siRNA polyplexes but not with the inhibitors, were used as positive controls to which it was attributed the value of 100% fluorescence. 2.14. Statistical analysis P values were calculated by the GraphPad InStat tools (GraphPad Software, Inc., La Jolla, CA, USA) as appropriate. P values < 0.05 were considered to be statistically significant. 3. Results and discussion 3.1. Synthesis via EMS and characterization of Inu-DETA copolymer Organic reactions that combine features of high yields and simple execution conditions are currently seeing with interest. Among synthetic methods, the use of microwave irradiation has become increasingly popular within the polymer synthesis and modification area (Singha et al., 2012). This methodology offers significant advantages with regard to product yield, simplicity in operation and green aspects. Additionally, microwave irradiation requires significantly reduced reaction time compared to conventional heating approaches. While these last occur by convection and require a long time for the heating of the whole reaction flask, heating by microwave irradiation occurs faster and more homogeneously in the reaction flask. In particular, microwave irradiation can more rapidly and directly transfer the heat to the reaction molecules, thus allowing a larger percentage of molecules to get the required activation energy (Hayes and Collins, 2004). Here we have employed an alternative recent method to perform microwave-assisted organic reactions, termed EMS (Hayes and Collins, 2004). With the simultaneous administration of microwave irradiation and the external cooling of the reaction vessel, more energy can be directly applied to the reaction mixture, thus ensuring a high and constant energy level at a constant reaction temperature. This method, as also experienced by us in our previous works (Sardo et al., 2014; Cavallaro et al., 2009; Scialabba et al., 2014), allows to easily functionalize linear polymers with oligoamine at a higher efficiency compared to conventional thermal approaches. Additionally, EMS, ensures a substantial time and reagents saving also limiting the occurrence of slower side reactions. Inu-DETA synthesis (Fig. 1A) has been conducted in an overall time of 5 h subdivided into 1 h of activation of inulin hydroxyls with Bis(4-nitrophenyl) carbonate (BNPC) by EMS, under controlled conditions (power, temperature and time) and 4 h of

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reaction in the presence of DETA. The addition of activated inulin to DETA solution in a-DMF has been performed in 40 min, to avoid formation of reticulated and insoluble sub-products. This last aspect was demonstrated by the complete solubility of the obtained Inu-DETA in water before the GPC purification step and by the absence of signals in the DLS analysis attributable to polymer aggregates scattering (Fig. 2C). The pure product was characterized by 1H NMR analysis to confirm the attributed structure. 1H NMR spectrum of Inu-DETA (Fig. 1B), shows additional peaks compared to those found in the inulin spectrum. In particular, three peaks are visible at the chemical shift comprised between d 2.7 and d 3.2 ppm (2.7 (m, 4HDeta, – CH2–NH–CH2–), 2.8 (m, 2HDeta, –CH2–NH2), 3.2 (m, 2HDeta, –O– CO–NH–CH2). The functionalization extent in terms of derivatization degree (D.D. mol%), defined as the percentage of the molar ratio between DETA and the fructose repeating units of inulin, was calculated as the ratio between the area of the signals corresponding to methylenic groups of DETA (at d 2.7–3.2 ppm), and that of the fructose repeating units of inulin (at d 3.5–4.5 ppm). The D.D. mol% resulted to be equal to 28 ± 2 mol% while the targeted value was of 50 mol% (mmol BNPC/mmol fructose repeating units = 0.5)’’. The average molecular weights of inulin and of the synthesized Inu-DETA polymer were determined by performing SEC analysis in acidic aqueous media. While inulin Mw was 5218, with a polydispersity index (PDI = Mw/Mn) of 1.46, for Inu-DETA a value of 5913 Da was found with a PDI of 1.31 (Table 1). 3.2. Characterization of Inu-DETA/siRNA complexes The structure of Inu-DETA contains 1,2-diaminoethane moieties grafted onto inulin backbone to confer the polymer the characteristic to possess positive charges at physiological pH. Diethylenetriamine in fact has two primary amine groups with a pKa between 9 and 10; in our copolymer one of the two amine groups is bound to inulin hydroxyls to form an urethane bond while the second amine group, with a pKa reported to be of 4.78 (Montemayor, 2008), is free. To determine whether the diethylenetriamine present in Inu-DETA can electrostatically bind negatively charged siRNA molecules, an electrophoresis analysis on agarose gel was performed. As reported in Fig. 2A, Inu-DETA was able to significantly arrest the electrophoresis run of siRNA molecules starting from a polymer/siRNA weight ratio of 7. This is evidenced by the band present on the bottom of the gel of Fig. 2A for lanes R7R40. Although reduced in extent, at lower ratios (Supplementary material 1), the retardation of siRNA migration is still visible, suggestive of a somewhat weaker interaction polymer/siRNA. This is evidenced by the smear observable in lanes R2-R5 (Fig. 2A and Supplementary material 1). To achieve an efficient siRNA distribution throughout the cytoplasm, the chosen delivery system has to possess specific properties correlated to its surface charge, size and shape. Rejman et al. showed that the size and nature of the delivery system influence the internalization mechanism (Rejman et al., 2004) and that differences in nucleic acid release influence the transfection efficiency (Rejman et al., 2005). In contrast, Spagnou et al. did not observe major differences in gene silencing using different sizes of siRNA loaded particles (50–100 nm or 200–600 nm) in vitro (Spagnou et al., 2004). Together these findings suggest that the factors influencing siRNA delivery are yet poorly understood. Thus, a characterization of size, shape, surface charge, polymer nature and charge density should be carefully investigated for any novel siRNA delivery system. To correlate the Inu-DETA/siRNA effects in terms of uptake and silencing with the physico-chemical properties of Inu-DETA/siRNA polyplexes, we started with the determination of the size and zeta potential by means of DLS technique, in DPBS at pH 7.4. Inu-DETA

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C. Sardo et al. / European Journal of Pharmaceutical Sciences 75 (2015) 60–71

(A) Schematic representation of Inu-DETA synthesis. OH O

OH O

OH OH

HO O

OH OH

HO O

OH

1)

OH

O

NO2

O

2)

OH O

OH O

OH H 2C OH

n

OH

H2 C

O

OH

O

OH

H 2C

O

OH

NO2

O O

H N

H2N

NH2

N H

O

OH H 2C

H N

O

O

OH O

NH2

OH H2 C

DMF

n

OH

O

1) 1h, 25W, 60°C

OH O

2) 4h, RT

OH H2 C OH

OH

H N

O

NH2

N H

O

(B) Representative 1H NMR spectrum of Inu-DETA in D 2O. OH O

OH OH

HO O

d

OH

d H2C O

d

a

H N

O

O

c

O

a N H

NH2

b

e OH

f

OH O OH

H2C

OH

d

O OH H2C OH

f

n

OH

O

H N

O O

e

N H

NH2

a c

b

Fig. 1. Scheme of Inu-DETA synthesis and 1H NMR spectrum. (A) Schematic representation of Inu-DETA synthesis. (B) Representative 1H NMR spectrum of Inu-DETA in D2O.

formed complexes with siRNA molecules at all the weight ratios (R) tested (Fig. 2B). For R lower than 10, polyplexes exhibited a large size, up to 700 nm, with a high polydispersity evidenced by the high PDI values, thus suggesting an aggregation phenomena. From R10 on, polyplexes exhibited smaller sizes (range 300– 400 nm) with an excess of polymer in solution not associated with siRNA, as shown in the left panel of Fig. 2C. Here, the graph obtained by DLS analysis of polyplexes at R equal to 40 shows that two different species are present, in the range between 1–10 nm and 100–600 nm, attributable to Inu-DETA not associated with siRNA and polyplexes, respectively. This is confirmed by DLS data recorded for Inu-DETA alone and siGL2 alone, reported in the right panel of Fig. 2C. Increasing the weight amount of copolymer in the polyplex formation, the Zeta potential values increased from 30 mV for the naked siRNA molecules to progressively more positive values, reaching the positivity around R7 (Fig. 2B). These results are in good agreement with the agarose electrophoresis assay where the switch between fully positive (complete siRNA migration inhibition) and negative/weakly positive particles (partial siRNA

migration inhibition) occurred at R7. Additionally this observation is in line with the size distribution obtained with DLS. Here, near to the region of Zeta potential neutrality, the repulsion forces among nano-complexes are weak thus justifying the aggregation phenomena, witnessed by the higher polyplex size and PDI values we detected. Even if many methods (such as DLS, AFM, NTA, FCS, TEM) are suitable for the characterization of nanoparticle medium size and homogeneity, our samples characterized by a certain heterogeneity, are difficult to analyze by only one of these methods. Thus, DLS data were compared with a direct microscopic inspection, useful to analyze the heterogeneity of samples thus allowing a more refined interpretation of the results. TEM analysis of Inu-DETA/ siRNA polyplexes at various weight ratios, revealed spheroidal nanosized complexes, confirming also DLS measurements data. Representative TEM images of Inu-DETA/siRNA polyplexes at R = 20, recorded at various magnifications, are reported in Fig. 2D. In this case, the average diameter of polyplexes as extrapolated by Image J software, was of 245 ± 127 nm, in line with the data of Fig. 2B.

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Characterization of Inu-DETA polyplexes A

B

siRNA naked R5

R7

R10

R20 R30 R40

%intensities

C

Size (d.nm ) Inu-DETA/siRNA R40

siRNA

Inu-DETA

D

Fig. 2. Characterization of Inu-DETA polyplexes. (A) Agarose gel electrophoresis retardation assay of Inu-DETA/siRNA polyplexes at different weight ratios (R); (B) particle size (black indicators), PDI values (data labels) and Zeta-potential (red indicators) of Inu-DETA/siRNA polyplexes at various weight ratios in DPBS (pH 7.4) measured by DLS (data are reported as means ± SD, n = 3); (C) graph obtained by DLS analysis of polyplexes at R equal to 40 (left panel), Inu-DETA and naked siRNA (right panel); (D) Representative TEM images of polyplexes at R = 20. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3. Relative buffering capacity of Inu-DETA

Table 1 Molecular characteristics of INU-DETA copolymers. Properties of Inulin, Inu-DETA and DETA

a b c d

b

d

Compound

pKa

pH

Relative buffering capacityc

Mw (Mw/Mn)

Inulin Inu-DETA DETA

12,03 (fructose) / 9.94; 9.23; 4.78a

5.60 8.92 9.54

/ 53% 13%

5218 (1.46) 5913 (1.31) 103.17

In water at 25 °C. In water at 25 °C, at a concentration of 0.2 mg/ml. (DVHCl7.4–5.1  0.1 M)100/Nmol. Determined by aqueous SEC analysis (pullulan standards calibration).

The variation in the protonation degree between pH 7.4 of the extracellular/cytoplasmic environment and pH 5.1 of endosomes, seems to be a crucial factor for successful transfection with polycation-based systems (Kaneda, 2001; Brown et al., 2001). It is known that cationic polymers, which contain many secondary and tertiary amine moieties, facilitate endosome escape due to their buffering effects occurring between cytoplasmic and endosome pH (Varkouhi et al., 2011). This is the case of the well-known polyethylenimine (PEI) and derivatives, which shows a high buffering profile in a wide pH range and a high transfection ability,

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(A) Analysis of the buffering capacity of Inu-DETA copolymer 12 10 8

Inu-DETA pH

DETA

6

Inu 4

7,4 5,1

2 0 0

200

400

600

800

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(B) Analysis of the stability of Inu-DETA/siRNA polyplexes

(I)

siRNA naked R5

R7

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siRNA naked R5

R7

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Fig. 3. Analysis of the buffering capacity of Inu-DETA copolymer and effects of albumin on complexes stability. (A) Acid base titration of Inulin (Inu), Inu-DETA, and DETA, at the same concentration present in Inu-DETA, against 0.1 M HCl. (B) Electrophoresis following 4 (I) and 8 h (II) of polyplexes incubation with albumin.

although their use appears to be problematically influenced by the cytotoxic effects. It has been previously reported (Miyata et al., 2008) that polymer derivatives bearing 1,2-diaminoethane side chains exhibit a peculiar two-step protonation behavior that facilitates membrane destabilization at the acidic pH of late endosome or lysosome. These polymers maintain a good stability profile with regard to the other cytoplasmic membranes at pH 7.4; in addition they possess low cytotoxicity and high transfection efficacy compared to the commercially available linear PEI and lipid based systems. Based on the above considerations, we determined the buffering capacity of the Inu-DETA copolymer, conferred by the 1,2-diaminoethane side chains. The acid-base titration profile was evaluated also for Inulin and DETA alone, used at the same concentration present in the Inu-DETA copolymer. While the titration curve of Inulin solution showed a rapid reduction in pH value (Fig. 3 and Table 1), suggesting a minimal buffering capacity, InuDETA titration profile showed a good buffering capacity. This is due to the DETA functionalization that confers a two-step protonation behavior. This behavior depends on the fact that the moles of protonated amines in the pH range 7.4 and 5.1 respect to the total moles of protonable amine groups, calculated as RBC7.4–5.1 (see Section 2.5), resulted to be around 50%.

Several factors could affect the dissociation of complexes such as the charge density and distribution in the structure of the polymeric carrier, the molecular weight of the copolymer and the possibility of ionic interaction with other charged bio-macromolecules (Merkel and Kissel, 2014). In particular, the serum abundant and negatively charged protein albumin can displace negatively charged molecules such as siRNAs from the positively charged carrier. Thus, we investigated the effects of Bovine Serum Albumin (BSA) on the Inu-DETA/siRNA polyplexes stability. After 4 or 8 h from the addition of albumin to polyplexes (final BSA concentration of 40 mg/ml) prepared at Inu-DETA/siRNA weight ratios ranging from 5 to 40, the stability was assessed by agarose gel electrophoresis. For R P 10 the incubation of Inu-DETA/siRNA polyplexes with BSA, did not lead to a different siRNA shift (Fig. 3B I) compared to the electrophoresis performed without BSA (Fig. 2A). This suggests that neither major destabilization nor polyanion exchange occurred. In contrast, for R 6 7 a more evident siRNA detachment from the polymer (as evidenced by the smear in the gel) was detected already after 4 h of incubation, compared to the electrophoresis performed without BSA (Fig. 2A). This observation indicates a certain destabilizing effect caused by BSA. The effect was even more evident following 8 h of incubation (Fig. 3B II). 3.5. Cytocompatibility studies

3.4. Stability of Inu-DETA polyplexes in the bloodstream Polyplexes stability in the bloodstream is of fundamental importance to avoid the premature release of the therapeutic cargo.

A number of polymeric delivery carriers have been suggested for improved intracellular delivery of siRNAs. Various kinds of cationic species can form nanosized polyelectrolyte complexes

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Cytotoxicity – 16HBE

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cytotoxicity depends also on the presence of different functional groups in the polymeric structure. In fact, for example, the incorporation of hydroxyl groups on PEI surface strongly reduced cell toxicity compared to unmodified PEI (Luo et al., 2011). In our case, we believe that a relatively low molecular weight of Inu-DETA, the nature of linked protonable groups, their density (about 10 DETA molecules for chain) and the other different groups present in the polymeric structure (including hydroxyl groups) can be responsible for the high cyto-compatibility. Notably, the addition of the siRNA molecules (of irrelevant sequence) to Inu-DETA, did not affect the cyto-compatibility (Supplementary materials 2A and B), reinforcing the concept of the appropriateness our InuDETA/siRNA delivery device. 3.6. Silencing efficacy of the Inu-DETA/siRNA polyplexes

Cytotoxicity – JHH6

(B)

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25

50

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Inu-DETA (µg/ml) Fig. 4. Viability effects of Inu-DETA on 16 HBE and JHH6. (A) 16 HBE cells were treated by various concentrations of Inu-DETA for 4, 24 and 48 h; the data, expressed as% of non-treated cells (control), are reported as means ± SD, n = 6. (B) JHH6 cells were treated by various concentrations of Inu-DETA for 4, 24 and 48 h; the data, expressed as% of non-treated cells (control), are reported as means ± SD, n = 6.

with negatively charged siRNAs by ionic interactions. The resulting complexes can provide excellent protection of siRNAs, prolonged circulation properties and biodegradability. However, the administration of the polycation/siRNA complexes in vivo can result in their dissociation leading to the interactions of the noncomplexed polycations with blood components, extracellular matrix proteins and cell membranes, thus resulting in toxic effects (Fischer et al., 2003). We thus investigated this aspect for our polymer starting from the worst scenario, i.e. the one in which a complete dissociation of the Inu-DETA/siRNAs occurs. To this purpose, Inu-DETA polymer alone was incubated with two model cell lines, JHH6 and 16HBE. The reason for the use of these cell lines, is based on the fact that JHH6 are a human carcinoma cell line (hepatocellular carcinoma) while 16HBE are a non-tumor cell line (human bronchial epithelial). This difference gave us the possibility to compare the toxicity effects behavior in tumor vs non-tumor cells. In the concentration range of 25 lg/ml to 1000 lg/ml and after 4, 24 and 48 h, non-significant toxicity was observed as evaluated by studying cell viability (Fig. 4). Remarkable is the lack of toxicity also at the highest concentration tested (1000 lg/ml), considering that polycations are usually tested for in vitro cytocompatibility at concentrations not superior to 200 lg/ml. Our finding suggests that the major variables determining polymer cytotoxicity, i.e. the molecular weight, structure and the type of cationic moieties and conformational flexibility (Fischer et al., 2003), are, in the tested copolymer, optimal for the model cell lines considered. We believe it is particularly relevant the proper choice of the cationic moieties types. Indeed, whereas positive charges facilitate endocytosis through interactions with the negatively charged cell surface, they can also damage the cell membrane causing significant toxicity (Fischer et al., 1999). Moreover,

The effectiveness of the Inu-DETA/siRNA polyplexes was evaluated in the model cell lines used for the toxicity studies, i.e. JHH6 and 16HBE. The siRNA, previously selected by us (Farra et al., 2011), is directed against the transcription factor E2F1 (siE2F1), a major promoter of cell proliferation. Three days after the administration of Inu-DETA/siE2F1 (200nM, weight ratio 4:1, R4), we observed in JHH6 a significant reduction of E2F1 protein (Fig. 5A) and mRNA (Fig. 5B) levels compared to a control siRNA (Inu-DETA/siGL2). Additionally, also the mRNA levels of three genes transcriptionally controlled by E2F1, i.e. E2F3, cyclin D1 and cyclin A2 (Fig. 5C), were reduced. This observation fully supports a successful and functional depletion of E2F1 as further indicated by the significant reduction of the cell number (Fig. 5D). Optimal weight ratio for Inu-DETA/siE2F1 effect was found to correspond to R4. At this ratio the overall charge of the particles is negative (Fig. 2B), a fact which in principle may not be considered optimal due to the negatively charged surface of the cell membrane. However, previously (Grassi et al., 2007; Cavallaro et al., 2014) we observed that siRNA-carrying liposome/polymeric particles displayed optimal transfection efficacy of siRNAs in different cell types, despite having a negative surface charge in the range of that observed here. Thus, our present and past findings suggest that, at least in some cell types, a mild negativity of the surface of the carrier particle is favorable for siRNA delivery. In contrast to the encouraging effects of Inu-DETA/siE2F1 in JHH6, in 16HBE no significant activity of the siE2F1 was detected. This contrasting result prompted us to explore whether problems of Inu-DETA/siE2F1 uptake/trafficking could have been the reason for the failure of siE2F1 in 16HBE. 3.7. Cellular uptake of Inu-DETA/siRNA polyplexes Inu-DETA/siGL2 uptake studies in JHH6 and 16HBE cells were conducted using a fluorescently labeled siRNA (siGL2-Cy5). As at the weight ratio of 4 (R4) we got optimal functional results with Inu-DETA/siE2F1 in JHH6, we focused the uptake studies on R4 and on the nearest values of R2 and R7. Following 4 h of incubation and a subsequent extensive washing with DPBS to eliminate membrane bounded complexes, in both cell lines uptake was studied quantitatively (Fig. 6A and B), by measuring fluorescence intensity in cell lysates. A qualitatively evaluation (Fig. 6C and D) was also performed by fluorescence microscope analysis. Both cell lines were able to taken up polyplexes with the amount of internalized siRNA in 16HBE cells being lower than in JHH6 cells (compare Fig. 6A with B). 3.8. Determination of endocytosis pathways Whereas in16HBE the uptake studies revealed a spotted fluorescence (Fig. 6C), in JHH6 fluorescence was evenly distributed in

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(A) E2F1 protein level E2F1 GAPDH 0,6

1,2

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non treated cells

% normalized to

120 100

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Inu-DETA/siE2F1 R4

Inu-DETA/siGL2 R4

Fig. 5. Inu-DETA/siE2F1 functional effects. Inu-DETA was used to deliver siE2F1 (200 nM) to JHH6; three days after the administration, a significant reduction in the amount of E2F1 protein (A), mRNA (B) and cell number (D) were observed. Blot quantifications (A) were performed normalizing E2F1 bands to the respective GAPDH; the values were then expressed as fold change respect to siGL2-treated cells. E2F1 depletion was paralleled by the reduction of the mRNA levels of E2F3, cyclin D1 and cyclin A2 (C), all genes transcriptionally regulated by E2F1. siGL2 = control siRNA, R4 = weight ratio Inu-DETA/siRNA 4:1. Data are reported as mean ± SEM, n = 6; § p < 0.04, ⁄ p < 0.045, + p < 0.03.

the cells (Fig. 6D). This observation suggests possible differences in the uptake/trafficking mechanisms for Inu-DETA/siRNA in the two cell lines. As it is accepted opinion that the route of entry of nanosystems into the cells is decisive for their final intracellular localization and for their potential application as nucleic acid based drugs delivery agents (Vercauteren et al., 2012), we studied the endocytic pathways involved in the cellular uptake of InuDETA/siRNA polyplexes by exclusion studies. To this purpose, we employed the chemical inhibitors WORT, PAO and M-b-CD which are able to specifically affect macropinocytosis, clatrin mediated endocytosis and lipid and cholesterol mediated endocytosis, respectively (Araki et al., 1996; Denora et al., 2013). Since the effect of the endocytosis inhibitors is dose-dependent, the vital cellular processes may be compromised at high concentrations thus leading to reduced cell viability and changes in uptake pathways in a non-specific manner. Therefore, to obtain reliable results, we first optimized the inhibitors concentrations and incubation times for each molecule by evaluating the cytotoxic effects on the model cell lines. Based on these studies (Supplementary materials 3 and 4), 1 lM WORT, 1 lM PAO and 2 mg/ml M-b-CD were used for a pre-incubation time of 0.5, 1, and 0.5 h, respectively and a co-incubation time with complexes of 4 h for WORT and M-b-CD. PAO treated cells were subjected just to the pre-incubation period, because a 4 h co-incubation period

for both 16HBE and JHH6 cells with the inhibitor at a concentration of 1 lM, drastically reduced viability. Lower concentrations, either for pre- or co-incubation periods, were not effective in producing a stable inhibition. In analogy with the uptake studies above reported, also for the uptake inhibition investigations, we focused on the weight ratio Inu-DETA/siRNA of R4 and on the nearest values of R2 and R7. Cellular entry of Inu-DETA/siGL2-Cy5 polyplexes at all weight ratios considered in 16HBE cells seems to strongly depend on lipid and cholesterol mediated pathways, that include caveolae mediated endocytosis, since the presence of M-b-CD quantitatively reduces polyplexes uptake (Fig. 7A and C). Caveolae mediated pathway does not involve the acidification of the vesicles where the endocitated material is retained (Xiang et al., 2012). This lead us to conclude that membrane destabilization promoted by second step protonation of grafted DETA does not occur in 16HBE and thus polyplexes are not promptly released into the cytoplasm of the cells where the siRNA should perform its interfering action. This hypothesis is in agreement with the fact that in 16HBE the fluorescence distribution appears to be dotted (Fig. 6), suggestive of an accumulation/sequestration of siRNA in defined cytoplasmic vesicles. Treatment with WORT and PAO, known inhibitor of macropinocytosis (Araki et al., 1996) and clatrin mediated endocytosis,

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(B) Quantitative uptake of INUDETA/siGL2 –Cy5 polyplexes in JHH6 cells.

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Fig. 6. Inu-DETA/siGL2Cy5 uptake. (A) Quantitative uptake of Inu-DETA/siGL2-Cy5 in 16HBE. (B) Quantitative uptake of Inu-DETA/siGL2-Cy5 in JHH6; siRNA = naked siRNACy5. Data are reported as mean ± SEM, n = 6; ⁄ p < 0.05. (C) Fluorescence microscopy images of 16 HBE and (D) JHH6 cells treated with Inu-DETA/siGL2 polyplexes at a representative weight ratio (R4) using a siGL2 concentration of 200 nM, compared to naked Cy5-labelled siGL2 (200 nM); evident is the different distribution of siGL2-Cy5 in the two cell types.

respectively, reveals that these two uptake mechanisms could largely contribute to the internalization of Inu-DETA/siRNA complexes in JHH6 (Fig. 7B and D). As both uptake mechanisms involve the acidification of the vesicles where the endocitated material is retained (El-Sayed and Harashima, 2013), we concluded that the membrane destabilization promoted by the second step protonation of grafted DETA occurs in JHH6 allowing the release of the polyplexes into the cytoplasm where the siRNA can perform its interfering action. This hypothesis is in agreement with the fact that in JHH6 the fluorescence distribution appears to be even (Fig. 6), suggestive of a uniform distribution of siRNA in the cytoplasmic thus allowing the effective interaction with the target mRNA. In addition to this possibility, we believe the trafficking story in JHH6 may be even more complex. Indeed, despite being the Inu-DETA/siRNA polyplexes internalized with similar efficacies at the different weight ratios tested (R2, R4 and R7), we observed a clear functional effects of siE2F1 only at R4 (Fig. 5). We think this may depend on the interaction strength between Inu-DETA and the siRNA. Indeed, the gel electrophoresis retardation assay (Fig. 2A) indicates for R P 7 a relevant retardation effect on the siRNA migration, i.e a strong interaction Inu-DETA/siRNA. For R < 4 (Supplementary data 1) retardation is reduced thus suggesting a weak Inu-DETA/siRNA interaction. We thus hypothesize that at R7, despite the siRNA can be introduced into the cells, it is not efficiently released in the cytoplasm being too tightly bound to the Inu-DETA polymer. In contrast, for R < 4 the interaction is too weak and thus the siRNA may not be able to properly follow the Inu-DETA pathway, despite being up taken by the cells. Together, the above data indicate that both cell lines can uptake, although with somewhat different efficacy, the InuDETA/siRNA. Thus the lack of siE2F1 effect in 16HBE unlikely depends on an uptake problem. Instead, we believe it may be

ascribed to the different trafficking mechanism. In JHH6 but not in 16HBE, the polyplexes trafficking allows the free distribution of the siRNA in the cytoplasm thus permitting an efficient siRNA action. Whereas the reasons for the different trafficking in the two cell lines deserves further investigation, the present observation suggests a somewhat cell specific effects of the Inu-DETA/ siRNA polyplexes. The understanding of the reasons for the different trafficking mechanisms may allow to develop cell specific delivery polyplexes at the trafficking level based on Inu-DETA. 3.9. Conclusion The identification of simple, cost-effective and biocompatible materials is crucial for the development of effective siRNA delivery carriers. In this regard, the use of carriers based on cationic polymers has to deal with the well-known problem of cyto-compatibility. Indeed, if on one hand the positive charges mediate complexation and interaction with nucleic acids, on the other hand they are responsible for cell toxicity. Thus, the accurate determination of the optimal amount of positive charges is of utmost relevance. This is particularly true for the cationic polymers PEI that, although having high siRNA condensation and transfection ability, are not biodegradable and induce cellular death via necrosis and apoptosis in a variety of cells (Boeckle et al., 2004). To reduce the PEI induced cytotoxicity, a wide range of moieties were introduced to its structure (Oh and Park, 2009; Bruno, 2011) such as carboxyalkyl chains, PEG and other polymers/molecules. Whereas these modifications resulted in significantly reduced cytotoxicity, they induced a reduction in the neutralization capacity and siRNA condensation/stability. Based on these problems, many efforts have been pursued to develop delivery materials safe, biocompatible, able to properly interact with components of the

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Fluorescence intensity (A.U)/mg protein in %

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Fig. 7. Inu-DETA/siGL2Cy5 uptake in the presence of chemical endocytosis inhibitors. Quantitative uptake in 16HBE (A) and JHH6 cells (B) of Inu-DETA/siGL2-Cy5 polyplexes at representative weight ratios (R) in the presence of WORT, M-b-CD and PAO; also shown are correlated representative fluorescence microscopy images for 16HBE (C) and JHH6 (D). Results are shown as percentage of non-inhibited cells treated with polyplexes at the same R. Data are reported as means ± SD, n = 3; ⁄ p < 0.05 respect to nontreated cells (NT).

complex biological environment and easy to chemically manipulate. In this contest, we believe the use of inulin derivatives represents a way to overcome the toxicity obstacles without the need of extensive chemical modification, otherwise required for polymers such as PEI. Our data clearly show the totally negligible toxicity of the Inu-DETA (Fig. 4) and the satisfactory transduction ability in JHH6 (Figs. 5 and 6B and D). In the present work we recurred to the novel technique of EMS to generate a novel siRNA delivery device based on inulin, an abundant and natural polysaccharide considered to be biocompatible and tunable for the specific requirements of siRNA delivery. Bound to inulin, there are diethylenetriamine residues which, due the positive charges, can complex the negatively charged siRNAs. The results here presented indicate that Inu-DETA polyplexes can effectively bind siRNAs, are highly cyto-compatible and, in the hepatocellular carcinoma cells JHH6, can effectively deliver functional siRNA targeted against the mRNA of the

transcription factor E2F1. Optimal delivery is observed using a weight ratio Inu-DETA/siRNA of 4 which corresponds to polyplexes with an average size of about 600 nm and a slightly negative charge when bound to siRNAs. Moreover, we show that the uptake and trafficking mechanism, mainly depending on micropinocytosis and clatrin mediated endocytosis, allows the even distribution of siRNA within the cytoplasm of JHH6 at R4, thus justifying the functional effects observed. Notably, in 16HBE where the trafficking mechanism (caveolae mediated endocytosis) does not allow an even distribution of siRNA within the cell cytoplasm, no significant effects are observed. This data stresses the relevance of the proper trafficking for efficient siRNA delivery and suggests that delivery specificity may be also achieved at the trafficking level. In conclusion, we have developed a novel Inulin/based siRNA delivery system which can release siRNAs in a cell line of hepatic origin with good efficacy and substantially no cytotoxicity. Further studies in different cell lines and in in vivo models will

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contribute to fully define the delivery potential and features of our Inu/DETA polyplexes. Acknowledgements This work was in part supported by the ‘‘Fondazione Cassa di Risparmio of Trieste’’, by the ‘‘Fondazione Benefica Kathleen Foreman Casali of Trieste’’, by the ‘‘Beneficentia Stiftung’’ of Vaduz Liechtenstein and by the Italian Minister of Instruction, University and Research (MIUR), PRIN 2010-11, [20109PLMH2]. TEM experimental data were provided by Centro Grandi Apparecchiature – UniNetLab – Università di Palermo funded by P.O.R. Sicilia 2000–2006, Misura 3.15 Quota Regionale. The authors declare that there is no conflict of interest. Appendix A. Supplementary materials Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejps.2015.03.021. Reference Agostini, F., Dapas, B., Farra, R., Grassi, M., Racchi, G., Klingel, K., Kandolf, R., Heidenreich, O., Mercatahnti, A., Rainaldi, G., Altamura, F., Guarnieri, G., Grassi, G., 2006. Potential applications of small interfering RNAs in the cardiovascular field. Drug Future 31, 513–525. Araki, N., Johnson, M.T., Swanson, J.A., 1996. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol. 135, 1249–1260. Baiz, D., Dapas, B., Farra, R., Scaggiante, B., Pozzato, G., Zanconati, F., Fiotti, N., Consoloni, L., Chiaretti, S., Grassi, G., 2014. Bortezomib effect on E2F and cyclin family members in human hepatocellular carcinoma cell lines. World J. Gastroenterol. 20, 795–803. Ballarin-Gonzalez, B., Howard, K.A., 2012. Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effects and solutions. Adv. Drug Deliv. Rev. 64, 1717–1729. Boeckle, S., von, G.K., van der Piepen, S., Culmsee, C., Wagner, E., Ogris, M., 2004. Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J. Gene Med. 6, 1102–1111. Brown, M.D., Schatzlein, A.G., Uchegbu, I.F., 2001. Gene delivery with synthetic (non viral) carriers. Int. J. Pharm. 229, 1–21. Bruno, K., 2011. Using drug-excipient interactions for siRNA delivery. Adv. Drug Deliv. Rev. 63, 1210–1226. Cavallaro, G., Licciardi, M., Amato, G., Sardo, C., Giammona, G., Farra, R., Dapas, B., Grassi, M., Grassi, G., 2014. Synthesis and characterization of polyaspartamide copolymers obtained by ATRP for nucleic acid delivery. Int. J. Pharm. 466, 246– 257. Cavallaro, G., Licciardi, M., Scire, S., Giammona, G., 2009. Microwave-assisted synthesis of PHEA-oligoamine copolymers as potential gene delivery systems. Nanomedicine (Lond) 4, 291–303. Dapas, B., Farra, R., Grassi, M., Giansante, C., Fiotti, N., Uxa, L., Rainaldi, G., Mercatanti, A., Colombatti, A., Spessotto, P., Lacovich, V., Guarnieri, G., Grassi, G., 2009. Role of E2F1-cyclin E1-cyclin E2 circuit in human coronary smooth muscle cell proliferation and therapeutic potential of its downregulation by siRNAs. Mol. Med. 15, 297–306. Deng, Y., Wang, C.C., Choy, K.W., Du, Q., Chen, J., Wang, Q., Li, L., Chung, T.K., Tang, T., 2014. Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies. Gene 538, 217–227. Denora, N., Laquintana, V., Lopalco, A., Iacobazzi, R.M., Lopedota, A., Cutrignelli, A., Iacobellis, G., Annese, C., Cascione, M., Leporatti, S., Franco, M., 2013. In vitro targeting and imaging the translocator protein TSPO 18-kDa through G(4)PAMAM-FITC labeled dendrimer. J. Control. Release 172, 1111–1125. El-Sayed, A., Harashima, H., 2013. Endocytosis of gene delivery vectors: from clathrin-dependent to lipid raft-mediated endocytosis. Mol. Ther. 21, 1118– 1130. Farra, R., Dapas, B., Pozzato, G., Giansante, C., Heidenreich, O., Uxa, L., Zennaro, C., Guarnieri, G., Grassi, G., 2010. Serum response factor depletion affects the proliferation of the hepatocellular carcinoma cells HepG2 and JHH6. Biochimie 92, 455–463. Farra, R., Dapas, B., Pozzato, G., Scaggiante, B., Agostini, F., Zennaro, C., Grassi, M., Rosso, N., Giansante, C., Fiotti, N., Grassi, G., 2011. Effects of E2F1-cyclin E1–E2 circuit down regulation in hepatocellular carcinoma cells. Dig. Liver Dis. 43, 1006–1014.

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