Improvements in Rational Design Strategies of Inulin Derivative

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IMPROVEMENTS IN RATIONAL DESIGN STRATEGIES OF INULIN DERIVATIVE POLYCATION FOR SIRNA DELIVERY Carla Sardo, Emmanuela Fabiola Craparo, Barbara Porsio, Gaetano Giammona, and Gennara Cavallaro Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00281 • Publication Date (Web): 29 May 2016 Downloaded from http://pubs.acs.org on June 1, 2016

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IMPROVEMENTS IN RATIONAL DESIGN STRATEGIES OF INULIN DERIVATIVE POLYCATION FOR siRNA DELIVERY AUTHOR NAMES. Carla Sardo, Emanuela Fabiola Craparo, Barbara Porsio, Gaetano Giammona, Gennara Cavallaro.

Lab of Biocompatible Polymers, Dipartimento di Scienze e Tecnologie Biologiche, Chimiche, Farmaceutiche (STEBICEF), University of Palermo, via Archirafi 32, Palermo 90123, Italy.

ABSTRACT. The advances of short interfering RNA (siRNA) mediated therapy provide a powerful option for the treatment of many diseases, including cancer, by silencing the expression of targeted genes involved in the progression of the pathology. On this regard, a new pH-responsive polycation derived from inulin, Inulin-g-imidazole-g-diethylenetriamine (INU-IMI-DETA), was designed and employed to produce INU-IMI-DETA/siRNA “Inulin COmplex Nanoaggregates” (ICONs). The experimental results showed that INU-IMI-DETA exhibited strong cationic characteristics and high solubility in the pH range 3-5 and self aggregation triggered by pH increase and physiological salt concentration. INU-IMI-DETA showed as well an high buffering capacity in the endosomal pH range of 7.4-5.1. In the concentration range between 25 and 1000 µg/ml INU-IMI-DETA had no cytotoxic effect on breast cancer cells (MCF7) and no lytic effect on human red blood cells. ICONs were prepared by two-step procedure involving complexation and precipitation into DPBS buffer (pH 7.4) to produce siRNA loaded nanoaggregates with minimized surface charge and suitable size for parenteral administration. Bafilomycin A1 inhibited transfection on MCF-7 cells, indicating that the protonation of the imidazole groups in the endolysosome pathway favours the escape of the system from endolysosomal compartment, increasing the amount of siRNA that can reach the cytoplasm.

KEYWORDS. Inulin, siRNA, Diethylenetriamine, Imidazole, MCF-7.

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INTRODUCTION. siRNA-based therapeutics hold great potential for treatment of various cancers by targeting signalling pathways and oncogenes that promote cell proliferation, cell cycle progression, invasion/metastasis and resistance mechanisms in tumors. However, many challenges, including siRNA rapid nuclease degradation, poor cellular uptake and off-target effects, need to be addressed in order to carry these molecules into clinical trials 1. Therefore a delivery vector is required, and nano-technologies based on polyelectrolyte complexes seem to be a good approach in order to overcome these limitations, obtaining both protection against degradation and improvements in terms of cellular uptake. Most frequently, siRNA is condensed with a high charge excess of cationic polymer in aqueous solutions with low ionic strength, forming stable particles of submicron size with positive ζ-potential. If on the one hand, the positive surface charge in water prevents particle aggregation because of electrostatic repulsion, on the other hand it allows self aggregation triggered by physiological salts and serum components, vessel endothelium and blood cells following intravenous injection. This aspects of colloidal stability in isotonic saline buffers and in the presence of blood components hamper the in vivo application of siRNA nanotherapeutics. During the last years, our group investigated on the optimization of different polymer-based nanosystems for siRNA delivery and targeting employing different methods

2,3

and starting from high biocompatible

polymers such as the polysaccharide inulin. Inulin is a unique fructan-type polysaccharide, that consists of (2→1) linked β-d-fructosyl residues, usually with an (1↔2) α-d-glucose end group. The high biocompatibility, flexibility and the presence of three hydroxyls per fructose repeating unit make this material an excellent candidate for designing and producing technologically advanced systems for biologically active compounds4 5 delivery. Comparing with other polysaccharides, to date inulin has been insufficiently explored as starting biomaterial for the development of siRNA delivery systems 6. In spite of this, recently we showed that Inulin-Diethylenetriamine (Inu-DETA)-based 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 2 ACS Paragon Plus Environment

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delivery was 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 7. The aim of the present work is to provide improvements in the rational design strategy and synthesis of a inulin based polycation. For these reason, functional groups such as imidazole (IMI) and diethylenetriamine chains (DETA) were grafted onto inulin backbone. IMI is able to improve the buffering ability of final copolymer in the endosomal pH fall (pH 7.4→5.1) and to confer pH sensitivity to the final polymer and to the siRNA complex, while DETA is able to confer cationic properties to the complexing copolymer. This work also focused on the relevance of methods employed for the complexes formation and on the importance of dispersion media used for their preparation and characterization. The resulting copolymer, Inu-IMI-DETA, was then used to condense high amounts of siRNA molecules into nanoparticles, that will be internalized into target cells via endocytosis. In the cellular endosomes, these particles will respond to the pH decrease, which triggers endosomal membrane destabilization and cytoplasm release of encapsulated siRNA, that will be, finally, able to interact with specific intracellular targets and to produce the desired therapeutic activity.

EXPERIMENTAL SECTION Materials and methods Inulin from Dahlia Tubers (Mw ≈ 5000 Da), Diethylenetriamine (DETA) and 4-Imidazoleacetic acid hydrochloride (IMIAc), Bis(4-nitrophenyl) carbonate (4-BNPC), N,N′-Dicyclohexylcarbodiimide (DCC), 4-(Dimethylamino)pyridine (DMAP), dichloromethane (DCM), acetone, agarose, ethidium bromide, Ribonuclease A from bovine pancreas (RNase A) were purchased from Sigma Aldrich. Pullulan standards (in the range 180 - 47300 Da) were purchased from Polymer Laboratories. Anhydrous N,Ndimethylformamide (a-DMF) and were from VWR. Anhydrous Dimethylsulphoxyde (a-DMSO) was purchased from Alfa Aesar.


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H NMR spectra were recorded in D2O (VWR) using a Bruker AC-250 spectrometer operating at 250.13

MHz. FT-IR spectra were recorded on solid sample using a Bruker ALPHA FT-IR Spectrometer equipped with Eco ATR single reflection sampling module with a ZnSe ATR crystal. pH was recorded by using a pH-meter (HANNA HI 4221 equipped with an electrode HANNA HI1131). Organic solvents were evaporated by a evaporation system (Buchi) constituted by a waterbath B-480, a rotavapor R-114, a recirculating chiller F-105 and a vacuum controller V-800. siRNAs: Duplexed siRNA were purchased from eurofins MWG operon (Ebersberg). The gene target sequences (5’→3’) are reported herewith below: Luciferase GL3: CUUACGCUGAGUACUUCGA(dTdT), with and without Cy5 linked to the 5’ end of the sense strand

Cell culture Biological evaluations were conducted on human breast carcinoma cell lines stably transfected to express firefly luciferase gene (MCF-7/Luc, Cell Biolabs Inc. San Diego) or not (MCF-7, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna). MCF-7 and MCF-7/Luc were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum (FBS), 1% of glutamine 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.

General procedure for the synthesis and characterization of Inulin-g-Imidazole (INU-IMI) copolymer Two hundred mg of inulin (corresponding to 1.23 mmoles of fructose repeating units), previously dried in a oven at 70°C for 24 h, were dissolved in 2 ml of a-DMF. After complete dissolution a solution of IMIAc (200.7 mg, 1.23 mmoles in 3 ml of a-DMF) and a solution of coupling agents, DCC and DMAP, (305.7 mg DCC, 1.48 mmoles and 151 mg DMAP, 1.23 mmoles in 2 ml of a-DMF) were added and the mixture was kept under magnetic stirring at 25°C for 6h. The dicyclohexylurea formed was collected by 4 ACS Paragon Plus Environment

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centrifugation and the supernatant was precipitated in an excess of DCM. The obtained solid, collected by centrifugation (10000 rpm, 5 minutes, 5°C) was carefully dried under vacuum then dispersed in 10 ml of bi-distilled water, acidified until complete solubilization with 0.1 N HCl, and further purified by dialysis against water, using a Spectrapor Dialysis Tube with a molecular weight cut off of 100-500 Da. After purification the solution was freeze-dried obtaining a brown solid. The pure product was characterized by 1H NMR analysis in D2O/DCl. 1H NMR of INU-IMI reveals peaks at δ: 3.7 ppm (m, 5HInu, –CH2–OH; –CH–CH2–OH; –C–CH2–O–), 3.9 ppm (t, 1HInu, –CH–OH), d 4.1 ppm (d, 1HInu, –CH–OH), 4.3 ppm(m, 2HIMIAc –CH–CH2–O(CO)–CH2–C–), 7.3 ppm (m, 1HIMIAc, –CH– CH2–O(CO)–CH2–C=CH), 8.6 ppm (m, 1HIMIAc, –CH–CH2–O(CO)–CH2–C–N=CH–).

General

procedure

for

the

synthesis

and

characterization

of

Inulin-g-Imidazole-g-

Diethylenetriamine (INU-IMI-DETA) copolymer One hundred mg of INU-IMI (corresponding to 0.22 mmoles of free fructose repeating units) were dissolved in 3 ml of a-DMSO and, after the addition of 34 mg of 4-NPBC (corresponding to 0.11 mmoles) the mixture was placed in a CEM Discover Microwave Reactor. After 1h irradiation at 25W, keeping the temperature under 60°C by externally cooling the reaction vessel with compressed air, the reaction mixture was added drop wise and very slowly, to 0.5 ml of a 114 mg/ml DETA solution in aDMSO (corresponding to 0.55 mmoles of DETA). The reaction mixture was then kept at room temperature for 4 hours, under constant magnetic stirring, and then precipitated in an excess of acetone. The obtained suspension was centrifuged and the residue washed twice with the same solvent. The obtained solid was carefully dried under vacuum and then solubilized in distilled water pH 5 to be purified by dialysis against water, using a Spectrapor Dialysis Tube with a molecular weight cut off of 100-500 Da. After exhaustive purification the solution was freeze-dried. The pure product was characterized by 1H NMR analysis in D2O/DCl. 1

H NMR of INU-IMI-DETA reveals peaks at δ: 3.0-3.5 ppm (m, 4HDeta, –CH2–NH–CH2–; m, 2HDeta, –

CH2–NH2; m, 2HDeta, –O–CO–NH–CH2–), 3.7 ppm (m, 5HInu, –CH2–OH; –CH–CH2–OH; –C–CH2–O–), 5 ACS Paragon Plus Environment

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3.9 ppm (t, 1HInu, –CH–OH), d 4.1 ppm (d, 1HInu, –CH–OH), 4.3 ppm (m, 2HIMIAc –CH–CH2–O(CO)– CH2–C–), 7.3 ppm (m, 1HIMIAc, –CH–CH2–O(CO)–CH2–C=CH), 8.6 ppm (m, 1HIMIAc, –CH–CH2– O(CO)–CH2–C–N=CH–). Residual internal D2O (δ 4.8)

Size exclusion chromatography The weight-average molecular weights (Mws) and polydispersity (Mw/Mn) of INU, INU-IMI and INUIMI-DETA, were determined by aqueous SEC. The protocol involved two TSK gel column (G4000 PWXL and G3000 PWXL) from Tosoh Bioscience, connected to a Waters 2410 refractive-index detector. Phosphate buffer solution 0.1 M at pH 3.5 was used as the eluent at 35 °C with a flow rate of 0.6 ml/min. Pullulan standards (in the range 180 - 112000 Da) for calibration and samples were dispersed at a concentration of 2.5 mg/ml in the mobile phase, and injected after filtration with 0.45 µm cellulose acetate syringe filters.

Buffering ability of INU-IMI-DETA copolymer To determine the relative buffering capacity of INU-IMI-DETA copolymer, acid-base titration was performed. The samples were prepared by dispersing 10 mg of INU-IMI-DETA copolymer at a concentration of 1 mg/ml in 0.1 N NaCl. Initially, the pH was adjusted to nearly 10.0 with 0.1 N sodium hydroxide and then titrated by adding gradually 10 µl of 0.1 N HC1 until reaching a pH of 3. Titrations of unmodified inulin (1 mg/ml), DETA and IMI, at the same concentration present in INU-IMI-DETA, 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-IMI-DETA. The Relative Buffering Capacity (RBC7.4-5.1), defined as percentage of amine groups becoming protonated within the pH-range from 7.4 to 5.1, was calculated according to the following equation: RBC7.4-5.1 = (VHCl × 0.1M) · 100/Nmol 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 10 mg of INU-IMI-DETA copolymer. 6 ACS Paragon Plus Environment

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Measurement of size and zeta potential as a function of pH INU-IMI-DETA solution (1 mg/ml) in bi-distilled water was added of proper volume of 0.1 N NaOH or 0.1 N HCl solution, by a MPT-2 autotitrator (Malvern Instruments); size and ζ potential were recorded at 0.5 pH increment units using a Zetasizer Nano ZSP (Malvern Instruments).

Chemical hydrolysis Aliquots (20 mg) of INU-IMI-DETA copolymer were dispersed in PBS at pH 7.4 or 5.0 at a concentration of 4 mg/ml. Then, samples were incubated under continuous stirring (100 rpm) and after 1, 3, 7, 14 and 21 days each dispersion was purified by exhaustive dialysis (100-500 Da MWCO), and dispersions freeze-dried for further 1H NMR analysis.

Gel electrophoresis assay Twenty µl of complex samples, prepared by simple mixing of siRNA solution at the concentration of 0.1 mg/ml with the same volume of aqueous INU-IMI-DETA dispersion at various concentrations to obtain different INU-IMI-DETA to siRNA weight ratios, were loaded on a 1.5% agarose gel, containing 0.5 µg/ml ethidium bromide and run at 100 V in tris-acetate/EDTA (TAE) buffer pH 8 for 30 minutes. The gels were then visualized against an UV transilluminator and photographed using a digital camera.

Preparation of INU-IMI-DETA/siRNA “complex nanoaggregates” (ICONs) 25 µl of INU-IMI-DETA aqueous solutions (pH 4) at various concentration were mixed with 25 µl of siRNA aqueous stock solution (0.1 µg/µl) in order to obtain different INU-IMI-DETA/siRNA weight ratios. The mixtures were incubated at room temperature for 30 minutes to allow complex formation. The complex containing mixtures were than precipitated in 100 µl of HEPES buffer (20 mM, pH7.4) or DPBS (pH 7.4) to form ICONs.


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Size and ζ potential measurements Dynamic light scattering studies (DLS) were performed at 25 °C with a Malvern Zetasizer Nano ZSP instrument fitted with a 532 nm laser at a fixed scattering angle of 173°, using the Dispersion Technology Software 7.02. The intensity-average hydrodynamic diameter (nm), and polydispersity index (PDI) were obtained by cumulative analysis of the correlation function. Polyplexes containing solutions were then diluted with 400 µl of nuclease free DPBS and used to determine the Zeta potential. 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.

Atomic force microscopy (AFM) analysis For AFM, a drop (∼20 µl) of the ICONs dispersion (weight ratio 20) in bidistilled water at 0.1 mg/ml concentration was deposited onto freshly cleaved mica and allowed to dry freely in air, then observed with a Multimode V Nanoscope Veeco microscope, driven by a nanoscope controller.

Ethydium bromide (EtBr) exclusion assay Dose-dependent condensation and encapsulation efficiency of siRNA by INU-IMI-DETA was examined by the quenching of EtBr fluorescence in a EtBr exclusion assay. A fixed amount of siRNA (2.5 µg or 187 pmoles of siRNA in 25 µl of RNAse-free water) was mixed with increasing amounts of INU-IMIDETA contained in 25 µl of RNAse-free water pH 4, corresponding to 0-20 weight ratios of INU-IMIDETA/siRNA, and incubated at room temperature for 30 min. This was followed by complex precipitation in 100 µl DPBS (pH 7.4) and further incubation for 4 h. After this time 0.75 µg of EtBr in 50 µl of DPBS (pH 7.4) were added and the samples were further incubated for 15 min. The fluorescence was measured using a spectrofluorophotometer (Shimadzu RF-5301 PC) at an excitation wavelength of 544 nm and emission wavelength of 547 and 585 nm. The relative fluorescence intensities were


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calculated as the ratio between intensity at 585 nm and at 547 nm. Results were expressed as a percentage relative to the naked siRNA-EtBr sample.

Evaluation of resistance against RNase of siRNA loaded into ICONs To measure the ability of INU-IMI-DETA to protect siRNA against nuclease degradation the hyperchromic effect, i.e. the increase in absorbance at 260 nm that occurs when nucleic acids degrade, was used as previously described 8,9. ICONs (150 µl), prepared as described above, at various R (5 – 35) were diluted to a final volume of 0.65 ml with b.d. water and placed in a quartz cuvette. 25 ng of RNase A (70 U/mg) were then added in 5 µl, obtaining a final siRNA concentration of 286.97 nM. Enzymatic degradation was monitored for 20 minutes by measuring absorbance at 260 nm at various time intervals on a Shimazu UV-VIS spectrophotometer. Results were plotted as increment % of absorbance at 260 nm (Abs260) versus time (minutes). Each point of the obtained curves are presented as mean ± standard deviation for triplicated samples.

siRNA release study from ICONs Release profiles were determined by comparing the amount of released siRNA with the total amount of siRNA loaded into the ICONs as a function of time. The chosen method was the dialysis method: 3 ml of INU-IMI-DETA aqueous solutions (pH 4) at various concentration were mixed with 3 ml of siGL3-Cy5 aqueous stock solution (0.105 µg/µl) in order to obtain INU-IMI-DETA/siRNA weight ratios of 10, 20 and 30. The mixtures were incubated at room temperature for 30 minutes to allow complex formation. The complex containing mixtures were than precipitated in 12 ml of DPBS (pH 7.4) to form ICONs. After 1h the prepared dispersions were diluted with DPBS RNase free at pH 7.4 or 5 to a final volume of 20 ml and placed into cellulose ester dialysis membrane with a molecular weight cut off of 100 KDa (Bioteck CE Tubing Spectra/Por®Dialysis Membrane). Dialysis bags were immediately immersed in 200 ml of DPBS at pH 7.4 or 5 and incubated in an orbital shaker (37°C, 100 rpm). At scheduled time intervals, 25 ml of the external medium were withdrawn and replaced with clean DPBS RNase free. 9 ACS Paragon Plus Environment

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Samples were freeze dried and dispersed in 1 ml of nuclease free water prior to measure the fluorescence intensity by a Shimadzu RF-5301 PC spectrofluorophotometer (λex 649; λem 673). Data were corrected taking in account the dilution procedure. As control, the diffusion of the same amount of naked siGL3Cy5 from dialysis was monitored. To calibrate the spectrofluorophotometer and quantify the released siRNA, standards of different concentration of siGL3-Cy5 in the concentration range of 1000-10 nM were prepared by serial dilution in DPBS and analysed. Each experiment was carried out in triplicate and the results were in agreement within ± 5% standard error.

Measurement of size and zeta potential as a function of albumin concentration ICONs (10 ml) formed in DPBS at a INU-IMI-DETA/siRNA weight ratio equal to 20, at a siRNA concentration of 0.02 mg/ml, were added of aliquots of albumin solution in RNAse free bi-distilled water (5.5 mg/ml) until reaching a concentration of 2.5 mg/ml. Size and ζ potential were recorded by using a Zetasizer Nano ZSP equipped with a MPT-2 autotitrator (Malvern Instruments).

In vitro hemocompatibility Hemocompatibility assay was performed according with a previous reported procedure10. Human erythrocytes isolated from fresh citrated-treated blood were collected by centrifugation at 2200 rpm for 10 min at 4 C. The pellet was washed 8 times with DPBS at pH 7.4 by centrifugation and suspended in the same buffer. Afterwards, it was diluted in DPBS at pH 7.4 to a final concentration of 4 v% erythrocytes. This stock dispersion was used within 12 h after preparation. Two hundred microliters of INU-IMI, INUDETA or INU-IMI-DETA copolymer dispersions (in a concentration range between 25 and 1·103µg/ml) were added to the same volume of the erythrocyte suspension and incubated for 1h at 37 °C under constant shaking. After centrifugation, the release of haemoglobin was determined by photometric analysis of the supernatant at 540 nm. Complete haemolysis was achieved by using a 1 v% aqueous solution of Triton X-100 (100% control value). Each experiment was performed in triplicate and repeated twice. The erythrocyte lysis percentage was calculated according to the following formula: 10 ACS Paragon Plus Environment

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% lysis = (Abssample - Absblank) / (Abs100% lysis - Absblank) x 100 where Abssample is the absorbance value of the haemoglobin released from erythrocytes treated with INUIMI, INU-DETA or INU-IMI-DETA copolymers; Absblank is the absorbance value of the haemoglobin released from erythrocytes treated with DPBS buffer, and Abs100%

lysis

is the absorbance value of the

haemoglobin released from erythrocytes treated with 1 v% aqueous solution of Triton X-100. Results represent mean ± standard deviation for triplicated samples.

Cytocompatibility assay MCF-7 cells, were seeded in a 96 well plate at a density of 2·104 cells/well. After 24 hrs, 200 µl of fresh OPTI-MEM medium containing ICONs or INU-IMI-DETA alone were added to cells. Polyplexes, prepared as described above at weight ratios of 15, 20, 25 and 30, were diluted to a final volume of 200 µl with OPTI-MEM and added to cells, obtaining a ﬁnal siRNA concentration of 200 nM. INU-IMI-DETA polymer was incubated at various concentrations ranging from 25 to 1·103 µg/ml, while untreated cells were used as control with 100% viability. After 4, 24 and 48 hrs of incubation, cells were washed with 100 µl of sterile DPBS and incubated with fresh DMEM containing 20 v% of MTS reagent solution (3(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium). Plates were incubated at 37 °C for 2 h and after this time the absorbance of formazane was measured by a UV plate reader, at 492 nm. Wells filled with MTS reagents at the same concentration in DMEM were used as blank to calibrate the spectrophotometer to zero absorbance. The cell viability values (%) compared to control cells were calculated by (Abs sample / Abs control) ×100 and represent mean ± standard deviation for triplicated samples.

In vitro quantitative siRNA cellular uptake. To determine quantitatively the cellular uptake of ICONs-Cy5, MCF-7/Luc cells were seeded in a 24 well plate at a density of 1.2·105 cells/well. After 24h, the culture medium was replaced by 600 µl of OPTIMEM I medium containing ICONs-Cy5 at different weight ratios, to reach a final siRNA concentration of 11 ACS Paragon Plus Environment

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200 nM siRNA. After 4h and 24h incubation, cells were extensively washed with sterile DPBS and lysed in 100 µl lysis buffer (2% SDS, 1% Triton X-100, in sterile DPBS). The lysates were divided in two parts: the first one (75 µl) was used to measure the fluorescence intensity by a Shimadzu RF-5301 PC spectrofluorophotometer (λex: 647 nm; λem:673 nm) calibrated with standard solutions of siGL3-Cy5 at various concentration ranging from 10 to 1000 nM; the second one (25 µl) was used to evaluate the total protein amount by BCA protein assay. The results were expressed as the ratio ng siGL3 per milligram of protein and represent mean ± standard deviation for triplicated samples

In vitro qualitative evaluation of siRNA cellular uptake and intracellular localization: fluorescence microscopy MCF-7 cells were seeded in a 96 well pate at a density of 2·104 cells/well. After 24h the culture medium was replaced with 200 µl of OPTI-MEM I® medium containing ICONs prepared at various INU-IMIDETA/siGL3-Cy5 weight ratios, ranging from 5 to 35, and obtaining a final siRNA concentration per well of 200 nM. After 4h incubation cells were washed with 100 µl of sterile DPBS and fixed with 4% formaldehyde for 30 min. Subsequently, the formaldehyde solution was removed and the nuclei were stained with 100 µl of 4′,6-diamidino-2-phenylindole (DAPI) in DPBS at a concentration of 5·10-3 mg/ml. After 3 min incubation, DAPI solution was removed, the cells were washed three times with DPBS and observed by a Axio Vert.A1 fluorescence microscope (Zeiss). The images were recorded using an Axio Cam MRm (Zeiss). For intracellular localization studies cells were treated with 200 µl of OPTI-MEM I® medium containing ICONs prepared at INU-IMI-DETA/siGL3-Cy5 weight ratios equal to 10 or 20 and after 4h cell were washed with sterile DPBS and incubated with pre-warmed (37°C) 100 µl of complete DMEM containing LysoTracker® Green DND-26 fluorescent probe (final concentration 100 nM) under growth conditions to stain lysosomes. Then, cells were fixed, nuclei stained and observed by fluorescence microscope as reported above. Images were analysed by MetaMorph® Microscopy Automation & Image Analysis Software for colocalization study, by measure the area or integrated intensity of the two fluorescent 12 ACS Paragon Plus Environment

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probes (Cy5 and LysoTracker® Green DND-26) and also by measure how much of either probe does or does not overlap the other probe.

In vitro gene silencing Twenty four hours before experiment MCF-7/Luc cells were seeded in a 96 well plate at a density of 2·104 cells/well. The culture medium was then removed and replaced with 200 µl of medium containing ICONs prepared at various INU-IMI-DETA/siGL3 weight ratios, ranging from 5 to 35, and obtaining a final siRNA concentration per well of 200 nM. After 4, 24 or 48h of incubation cells were washed with sterile DPBS and incubated with fresh medium and lysed after further 24 h with 50 µl of LB 1X. In a parallel experiment, ICONs were co-incubated in 200 µl of OPTI-MEM with Bafilomycin A1 at a concentration of 200 nM. Luciferase gene expression was analyzed on 20 µl of lysates with the Luciferase Assay System (Promega), according to the product manual, using a GloMax 20/20 Luminometer (Promega). 25 µl of cell lysates were used to determine the total protein content by BCA protein determination assay. Transfection efficiencies of ICONs with various weight ratios were compared to each other and to naked siRNA as negative control. Results are shown as relative mean values of replicate of 3 ± standard deviation (% of cells with full luciferase expression) normalized by protein content (RLU/mg protein %). Transfection experiment by using ICONs containing a scrambled siRNA, which cannot target luciferase mRNA, instead of siGL3 have been also performed. In this case ICONs at R10, 20 and 30 containing scrambled siRNA were incubated with MCF-7/Luc cells for 24h, using a final siRNA concentration of 200 nM.


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RESULTS AND DISCUSSION. Synthesis and characterization of INU-IMI-DETA copolymer. Effective gene silencing requires specific delivery of siRNA into target tissue, cytosolic release in target cells and the membrane of endosomal-lysosomal compartment is the final barrier for effective siRNA delivery. pH-sensitive siRNA delivery systems have been designed to overcome this barrier and to facilitate cytosolic siRNA release utilizing the pH difference between plasma and endosomal-lysosomal compartments 11. The INU-IMI-DETA copolymer here investigated, was constructed starting from inulin, a natural polysaccharide, by grafting two different molecules: diethylenetriamine (DETA) and 4-imidazoleacetic acid hydrochloride (IMIAc). Grafting of DETA molecules lead to a copolymer bearing 1,2-diaminoethane moieties that, having a pKa between 8 and 10

12

, confers the characteristic of polycation to the final structure. Moreover, 1,2-

diaminoethane moieties exhibit a peculiar two-step protonation behaviour that facilitates membrane destabilization at the acidic pH of late endosome or lysosome by changing its conformation 13. The imidazole ring is the functional unit of the histidine amino acid and it has been reported to give significant improvement in transfection efficiency after grafting to various polymer backbone Polymers containing imdazole functionality, such as poly(histidine)

16

14, 15

, poly(4-vinyl imidazole)

.

17

,

chitosan-g-imidazole 18, Imidazolyl-PEI 19 and others imidazole containing polymers, peptides and lipids 16

have been considered for applications as tumor targeting drug delivery and non viral nucleic acids

delivery systems. Polymers containing imidazole groups, whose pKa is 6.0–6.5, are usually more cytocompatible respect to cationic starting polymers and can mediate endosomal escape through the hypothesized proton sponge effect, because they have buffering behaviour between pH 7 and pH 5.1. INU-IMI was synthesized by Steglich esterification, i.e. a mild reaction which allows the ester formation, in which the addition of DMAP is crucial for the efficient conjugation. Employing equimolar quantities of AcIMI and DMAP and a small excess of DCC respect to moles of repeating fructose units of inulin, a derivatization degree (DDmol%), defined as the percentage molar ratio between IMI and fructose repeating 14 ACS Paragon Plus Environment

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units of inulin, of about 50 ± 5 mol% was obtained. The extent of functionalization in terms of DDIMI mol% was calculated by 1H NMR analysis as the ratio between the area of the signals corresponding to imidazole ring protons (2H at δ 8.6 and 7.3 ppm), to the area of the signal corresponding to 1H of fructose repeating unit of inulin (at δ 4.2 ppm). INU-IMI was further functionalized with DETA molecules according with a synthetic protocol already used to obtain INU-DETA polycation 20, involving activation of free inulin hydroxyl groups with BNPC by Enhanced Microwave Synthesis (EMS). INU-DETA and INU-IMI-DETA synthesis are graphically represented in Scheme 1 and 2.

Scheme 1. Schematic representation of the synthesis of INU-IMI copolymer.


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Scheme 2. Schematic representation of the synthesis of INU-IMI-DETA copolymer.

The DDDETA mol% in, calculated as the ratio between the area of the signals corresponding to methylenic groups of DETA (at δ 2.5-3.3 ppm) to that of the fructose repeating units of inulin (at δ 3.5-4.5 ppm), was of 25 ± 3 mol% respect to moles of repeating fructose units of inulin. Representative 1H NMR spectra of INU-DETA and INU-IMI-DETA copolymers are reported in Figure 1. This means averagely that the number of imidazole molecules bounded per chain of inulin are twofold the number of DETA moieties and that for each inulin backbone, constituted averagely by 36 fructose repeating units, there are 18 IMI and 9 DETA grafted molecules.


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Figure 1. Representative 1H NMR spectra of IMIAc, INU-IMI and INU-IMI-DETA with DETA and IMI signals attribution.

The average molecular weights of INU-DETA and INU-IMI-DETA were determined by SEC analysis in acidic aqueous media: while INU-IMI Mw was 7858 Da, with a polydispersity (Mw/Mn) of 1.64, for INU-IMI-DETA a value of 8063 Da was found with a polydispersity of 1.73. Molecular characteristics of synthesized copolymers and starting inulin are reported in Table 1.


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Table 1. Molecular characteristics of INU, INU-IMI and INU-IMI-DETA copolymers

Polymer INU INU-IMI INU-IMI-DETA

Targeted DDmol% IMI

A

Sperimental DDmol% IMI

Targeted DDmol% DETA

/

/

/

100 %

50 %

/

40 %

A

Sperimental DDmol% DETA

BMw (Mw/Mn)

CRBC%

/

4782 (1.52)

/

/

/

7858 (1.64)

65 %

25 %

25 %

8063 (1.73)

56 %

by 1H NMR Determined by aqueous SEC RBC% : Relative Buffering Capacity % determined by acid/base titration

A Determined B C

To determine the buffering capacity of INU-IMI-DETA copolymer acid-base titration was performed. The titration proﬁle was obtained also for inulin, DETA and imidazole at the same concentration present in INU-IMI-DETA. While the titration curve of inulin solution showed rapid reduction in pH value, suggesting as expected no buffering capacity, INU-IMI-DETA titration profile show an high buffering as can be visualized by observing the slope of the curve in Figure 2. In fact the moles of protonated amines in the pH range between 7.4 and 5.1 respect to total moles of protonable amine groups, calculated as RBC7.4-5.1 as reported in the experimental section, resulted to be equal to 56% and 65% for INU-IMIDETA and Inu-IMI respectively. These values were both higher than that obtained for b-PEI25k, calculated equal to 27% 3. These results confirm the buffering ability of INU-IMI and INU-IMI-DETA copolymers in the pH interval from 7.4 (extracellular and cytoplasmatic pH) to 5.1 (endosomal/lysosomal pH).


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Figure 2. Acid base titration profiles (pH versus acid volume) of INU, INU-IMI and INU-IMI-DETA copolymers. Profiles of INU, INU-DETA, IMI and DETA and at the same concentration present in INUIMI and INU-IMI-DETA, were also reported.

With the aim to evaluate the eventual pH sensitivity of INU-IMI-DETA, copolymer dispersions were analyzed by DLS during acid/base titration, in term of size and zeta potential. The size and zeta potential profiles as function of pH, as reported in Figure 3, showed a pronounced pH-dependent phase transition and aggregation behavior of the copolymer around pH 7. This result can be explained considering that at low pH values, until about pH 6, the protonated form of imidazole and DETA residues are prevalent and this make inulin copolymer present in aqueous medium in small sized species, minor than 200 nm sized; on the contrary, increasing the pH values from 6 to about 7.5, the partial deprotonation of these group causes a decrease of hydrophilicity of copolymer and consequently the supramolecular aggregate formation characterized by a bigger size from 400 up to 1000 nm.


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Figure 3. Acid/base titration of aqueous INU-IMI-DETA dispersion with simultaneous size and zeta potential measurements by DLS.

Changing in the aggregation state was also observed together with variations in the ionic strength (IS) of the dispersion medium. In particular, measuring size of copolymer by DLS in various medium with the same pH (7.4) but different IS, i.e. bi-distilled water, HEPES (IS: 20 mM) and DPBS (IS: 200 mM), differences in size were observed; in effect, while in water an average hydrodynamic diameter up to 300 nm was observed, in DPBS, that have a ionic strength 10 fold higher than HEPES buffer, a pronounced aggregation occurred with an increase in size up to 900 nm. The size of the dispersion copolymer decreases after adding water, showing that the aggregation phenomenon was reversible (Figure 4). Moreover, the size of copolymer INU-IMI-DETA increases again if it is prior dispersed in bi-distilled water and then diluted with DPBS. This behavior can be explained considering that in the presence of a different ionic strength of the medium the ζ potential of dispersed colloidal species could decrease involving aggregation phenomena.


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Figure 4. INU-IMI-DETA size in various dispersion medium with different ionic strength (H2O: bidistilled water pH 7.4; HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid buffer 20 mM, pH 7.4; DPBS: Dulbecco’s Phosphate Buffered Saline pH 7.4; DPBS/H2O: samples prepared in DPBS and diluted with bidistilled water prior to analysis; H2O DIL DPBS: samples prepared in bidistilled water and diluted with DPBS prior to analysis).

Degradability of biomaterials is a very important issue for their use in medical application. To evaluate the degradability of chemical functionalization of INU by IMI and DETA grafting, a chemical stability study was done in a period of 21 days by incubating INU-IMI-DETA in DPBS at two different pH of 5 and 7.4. The degradation was followed by the determination of IMI and DETA DDmol% by 1H NMR analysis of INU-IMI-DETA at 1, 3, 7, 14 and 21 days, after dialysis purification of the dispersions in order to remove the formed water soluble hydrolysis products. Results in terms of DDIMImol% and DDDETAmol% are reported in Figure 5. As it can be seen in both media hydrolyses phenomena occurs leading to the DDIMImol% reduction. After 21 days at pH 7.4 a reduction of DDIMImol% value from 40% to 14.75% was detected and resulted to be significantly higher than that observed after 21 days of incubation at pH 5, when a reduction of DDIMImol% until about 25.5% was observed. The situation is quite different in the case of DETA portions where, as expected considering the stable nature of urethane linkage between DETA chain and hydroxyl group of inulin, no significative reduction of the DDDETAmol% was observed at day 21, for both pH conditions. 21 ACS Paragon Plus Environment

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Figure 5. Variations of DDIMI mol% (A) and DDDETA mol% (B) of INU-IMI-DETA dispersions in PBS at pH 5 and 7.4 as a function of incubation time. * represents significant difference with 0 incubation time point.

Preparation and characterization of INU-IMI-DETA/siRNA complex nanoaggregates (ICONs). To determine whether the INU-IMI-DETA can electrostatically bind negatively charged siRNA molecules, an electrophoresis analysis on agarose gel was performed. As reported in Figure 6, INU-IMIDETA was able to retard the electrophoresis run of siRNA molecules starting from a polymer/siRNA weight ratio of 10. This is evidenced by the band present on the bottom of the gel of Figure 6.

Figure 6. Agarose gel electrophoresis of INU-IMI-DETA/siRNA complexes obtained in water at various INU-IMI-DETA to siRNA weight ratios (R).


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With the scope to analyze copolymer properties upon interaction with siRNA, size and ζ potential of complexes formed by simple mixing of siRNA and copolymer dispersions, in the same dispersion medium (water, HEPES, DPBS), were measured by DLS. In bi-distilled water the formation of small, homogeneous and thigh complexes was observed with a size of about 100 nm (at polymer siRNA weight ratio between 5 and 35) with a positive ζ potential already starting from polymer-siRNA weight ratio of 5. This because in bi-distilled water at pH 7.4, a strong electrostatic interaction probably occurred between the cationic INU-IMI-DETA and the anionic siRNA, leading to the formation of tight positive complexes. On the contrary, in the presence of salts (HEPES or DPBS) larger complexes have been detected with size of about 500 nm in the case of HEPES and between 1000 and 1500 nm in the case of DPBS. In these conditions, counter ions weakened the electrostatic interactions by shielding the effective charge of copolymer and nucleic acids. Consequently, slightly charged polyplexes are formed, characterized by a smaller surface charge and aggregation phenomena (Figure 7).


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Figure 7. Size and ζ potential measured by DLS of complexes formed by simple mixing of siRNA and copolymer dispersions, in the same dispersion medium (H2O, HEPES and DPBS).

Starting from these considerations, to improve the formulation of a siRNA delivery system by employing INU-IMI-DETA copolymer, a new preparation method was developed, exploiting the strong pHdependent phase transitions and aggregation behaviour of INU-IMI-DETA copolymer. The new method involved a first step in which a certain amount of INU-IMI-DETA copolymer solution in water at pH 4.5 was mixed with the same volume of siRNA aqueous solution at defined polymer to siRNA weight ratios; at this stage, the copolymer is in the soluble form thanks to imidazole and DETA protonation, leading the copolymer to strongly interact with siRNA via electrostatic attraction to form complexes. Afterwards, the 24 ACS Paragon Plus Environment

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prepared copolymer/siRNA complexes were added into DPBS buffer (pH 7.4) to produce INU-IMIDETA/siRNA complex nanoaggregates (ICONs) via deprotonation of imidazole moieties. This approach lead to the formation of nanostructures with a diameter, determined by DLS, comprised between 200 and 350 nm for INU-IMI-DETA/siRNA weight ratios higher than 15 (Figure 8). Probably, being the complexation still not complete even at acidic pH for weight ratios under 15, complex nanoaggregates formed at lower weight ratios appear less stable and more polydisperse after precipitation into DPBS (400-1000 nm). This method allowed to stabilize polycation/siRNA interaction in the presence of salts and simultaneously to obtain tight complex nanoaggregates in a size range useful for parenteral administration in a physiological saline dispersion medium.

Figure 8. Size (black) and ζ potential (gray) of INU-IMI-DETA/siRNA complex nanoaggregates (ICONs) obtained by complexation/precipitation method at various weight ratios, measured by DLS.

The size and surface composition can strongly influence the biodistribution21 and the ability of nanoparticles to escape recognition and scavenging by the reticular endothelial system (RES), extravasate from the systemic circulation into tumor tissues, and become effectively internalized by target cells22. The ζ potential is a parameter which is considered by many authors as very important for the characterization of nanoparticles, but not only that: it is believed that zeta potential is one of the decisive factors in the adsorption of proteins on the surface of nanoparticles and it may also play a role in the interactions between the nanoparticles and the cells. Nanoparticles with a slightly negative charged surface can reduce 25 ACS Paragon Plus Environment

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the undesirable clearance by RES and improve the blood compatibility, thus deliver drugs more efficiently to the action sites23. While in DPBS dispersion medium naked siRNA showed a ζ potential of 12 mV, ICONs showed a ζ potential between -5 and 0 mV in the R range between 5 and 35. This ζ potential increase with increasing R, but never became positive, meaning that probably the high salt concentration shielded the electric charge of the complexes or that surface of ICONs is not completely covered by polycation. Moreover, the slightly charged surface of ICONS, almost in principle, allows the i.v. administration of these systems. Furthermore, to investigate the shape and surface morphology of the ICONs obtained with the complexation/precipitation method, AFM was used as visualization technique. This technique gave clear 3D morphological images (Figure 9), highlighting spheroid complex NPs with an average diameter of 103.33 ± 31.06 nm (see Figure S4), which is slightly smaller than the values determined by DLS in aqueous medium; this phenomenon, otherwise observed, is caused by the shrinkage of complex NPs upon drying 24, 25 and by the difference in the nature of the employed technique, being one a direct microscopy method (AFM) and the other an indirect method that take in account the hydration shell in aqueous dispersion.

Figure 9. Atomic Force Microscopy (AFM) of ICONs at R20.


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In addition to the role of buffering endosomes inside cells, imidazoles have been found to form hydrogenbonds with siRNA molecules. This is one of the key reasons why histidine-containing polyplexes are effective transfection and silencing agents

26

. Although alone these interaction do not lead to complex

formation, as experimented by means of electrophoresis of INU-IMI after incubation with siRNA at various weight ratios (data not shown), they could constitute an additional association force for siRNA complexation thus enhancing siRNA encapsulation efficiency and stability of the delivery system during circulation, avoiding premature release and degradation by nuclease of the therapeutic cargo. The trend of siRNA encapsulation efficiency was evaluated by determining indirectly the amount of siRNA, after ICONs formation, able to interact with ethydium bromide in an EtBr exclusion assay. As can be seen in Figure 10, the condensation of INU-IMI-DETA with the siRNA appear to be dose dependent and to reach the maximum condensation of 50% with weight ratio of 35.

Figure 10. siRNA encapsulation efficiency by ethydium bromide exclusion assay.

Once intravenously administered, prior to subsequent distribution to tissues and cells, nanoparticles circulate and interact with blood components, such as proteins, hydrolytic enzymes and cells. Interaction of nanoparticles with proteins gives rise to the formation of a dynamic nanoparticle-protein corona that may influence cellular uptake, inflammation, accumulation, degradation and clearance of the nanoparticles

27

. Understanding of such interactions can be directed towards generating biocompatible

nanomaterials with controlled surface characteristics in a biological environment. In the specific case of 27 ACS Paragon Plus Environment

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nucleic acid/polycation nanosystems, negatively charged proteins could also replace nucleic acid in the complex leading to a premature release of the therapeutic molecules. As consequence of protein corona formation or nucleic acid/protein exchange some parameters of complex nanoparticles change, such as hydrodynamic diameter and surface charge. Thus, to verify this possibility we have used two different techniques: DLS and electrophoresis on agarose gel after ICONs incubation with Bovine Serum Albumin (BSA). Albumin was chosen because of its abundance in human serum, for its negative-charged nature and because the interaction of complexes with albumin was proposed to have a great impact on the circulation of the complexes in blood. In fact, for example it has been shown that DNA-PLL complexes are rapidly cleared from the bloodstream, with a half-life of less than 5 minutes, and the reason of this brief circulation time reseed in the increase of complex size and modification of surface properties upon albumin interaction 28. DLS measurements revealed that the increasing concentration of albumin caused gradual, little change in ζ potential of ICONs (R 20) from ≈ -1 mV to more negative values, reaching constant ζ potential values around -8 mV above 1 mg/ml of albumin concentration, suggesting possible interaction of the complex nanoaggregates with albumin (Figure 11A). The measurement of the zeta potential was repeated after 4 h incubation with BSA at concentration of 40 mg/ml, that is in the physiological range29, confirming the value of -8.91 ± 1.55 mV. When BSA concentration reached 0.5 mg/ml, a second population of small particles with a hydrodynamic diameter around 9 nm was observed. We suppose that this population correspond to a small fraction of INU-IMI-DETA, that interact with albumin forming small INU-IMI-DETA/BSA complexes. This was observed also by Oupickỳ et al after treatment of Poly(HPMA)-block-poly(TMAEM)/DNA complexes with poly(styrenesulfonate)28. The small ζ potential variation until a constant slightly negative values (-9 mV) at a BSA concentration forty times lower than physiological concentration lead to suppose that interaction occurs with ICONs at R20 without any appreciable increment in size (Figure 11B). Although, zeta potential measurements alone cannot support speculations concerning the formation of protein corona on the surface of nanoaggregates. It is conceivable that this low interaction is due to the low and negative surface charge of complex


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nanoaggregates, that for all weight ratios in DPBS was measured, as dercribed above, between 0 and -5 mV. Electrophoresis after BSA treatment do not lead to appreciable variations in siRNA migration respect to untreated complexes used as control (Figure 11C), suggesting the absence of anionic polyelectrolyte exchange.

Figure 11. Interaction between INU-IMI-DETA/siGL3 complex nanoaggregates (R20) with Bovin Serum Albumin (BSA) determined by (A) ζ potential and (B) size measurements by DLS. (C) Electrophoresis on agarose gel after 4 h ICONs (R20) incubation with BSA at concentration of 40 mg/ml.


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One of the major barrier to siRNA bioactivity in vivo is degradation and inactivation by nuclease enzymes. Here, siRNA protection by ICONs was evaluated by the hyperchromic effect at 260 nm during exposure to RNase A. (Figure 12). The ICONs formulations effectively protected the siRNA against degradation. In fact, while after 20 min, free siRNA exposed to the RNase A showed a 20 % increase in Abs260, siRNA enclosed in ICONs exposed to the enzyme resulted in an increase in Abs260 up to 3.7 % for R 15. Starting from R 20 a little or no hyperchromic effect was observed reaching around 0 % for R 35. These data suggest that INU-IMI DETA protected siRNA from nuclease degradation for all weight ratios tested and starting from R 20, nanoaggregates appear to be highly efficient in the preservation of siRNA integrity respect to naked siRNA. Results are in agreement with encapsulation data, since weight ratios range that appear to be most efficient in encapsulating siRNA (R20-R35) is also the range the most efficient to protect it.

Figure 12. Evaluation of resistance against RNase of siRNA loaded into ICONs at INU-IMIDETA/siRNA weight ratios (R) in the range 5-35. R0 represent naked siRNA.

The in vitro release profiles of siRNA from ICONs in DPBS at a pH 7.4 showed a prolonged release with an almost linear pattern for up to 24h, and the total siRNA quantity released was relatively high, between ~60–85% of the initial loading quantity, for R10, 20 and 30 (Figure 13). Passing from R10 to higher R of 30 ACS Paragon Plus Environment

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20 and 30 there is a delay in siRNA release of 10%, being at 24h the amount of siRNA released respect to the loaded siRNA quantity of 84.6, 75.3 and 62.5% for R10, R20 and R30 respectively. This means that the release kinetic can be modulated by formulating ICONs with different amount of INU-IMI-DETA respect to siRNA. The same difference in the release behaviour was encountered when siRNA release was evaluated at pH 5. At this pH, in fact, at 24h the amount of siRNA released respect to the loaded siRNA quantity were found to be of 36.0, 23.3 and 6.7% respectively. Once ICONs are exposed to lower pH, i.e. pH 5, the release of siRNA from ICONs decreased consistently. This behaviour can be explained considering the difference of the zeta potential of the copolymer at these two pH values; in effect at pH 7.4 the copolymer has a ζ potential of ~ +10 mV, while at pH5 the ζ potential rises the value of ~ +30 mV, that could enhance the siRNA complexation ability of the copolymer leading to a decrease in the release profile. The excess of copolymer not directly involved in siRNA complexation is anyway more soluble at pH 5 and should be able to produce a proton sponge/destabilization effect mediated by imidazole and DETA moieties that permit the endo-lysosomal escape.

Figure 13. siRNA release profiles from ICONs in DPBS at pH7.4 (A) and pH5 (B), at INU-IMI-DETA to siGL3 weight ratios (R) of 10, 20 and 30. Diffusion of naked siRNA across membrane was followed for comparison.


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In vitro biological evaluations. Examination of the haemolytic potential is crucial to evaluating the toxicity of nanomaterials; here in vitro haemo-compatibility of INU-IMI-DETA, determined by haemoglobin release from red blood cells after incubation with the copolymer, was compared with INU-IMI and INU-DETA. It was observed that after 1h of incubation at 37°C, at the highest concentration of 1 mg/ml, the percentage haemolysis was slightly increased in comparison to control for INU-IMI and INU-IMI-DETA, reaching in the worst case 1% Haemoglobin release, while for the polycation INU-DETA no haemolysis was observed even at this high concentration, as shown in Figure S1 of supporting informations. According to the criterion in the ASTM E2524-08 standard

30

, percentage haemolysis > 5% indicates that the tested material causes

damage to red blood cells; however, this criterion was not exceeded at any of the tested concentrations, leading to the conclusion that INU-IMI-DETA could be potentially administered intravenously without red blood cell damage. We believe that our systems, having good biological performance when in contact with red blood cells, potentially shifted the risk/benefit ratio for siRNA delivery systems where the polymer amount can be substantially decreased. This potential advantage is corroborated by the cell cytocompatibility tests, which results are shown in Figure S2 of Supporting Information, where the cell viability is always over 85% respect to untreated cells, both for ICONs at copolymer/siRNA weight ratios in the range 15-30 and for INU-IMI-DETA alone in a concentration range up to 1 mg/ml, indicating a very good in vitro biocompatibility. Nevertheless, these exciting in vitro insights should be confirmed in vivo before generalized statements are made. The ability of ICON delivered siRNA to reduce the level of the target was assessed by a luciferase reporter gene knock down assay on MCF-7/Luc cells (Figure 14A). After 4h incubation, the transfection efficiency increases with R, reaching about a 35% reduction of the luciferase expression for R30. Interestingly, after 24h incubation the most effective ratio were R5 and 10 with a reduction of recovered luminescence up to 80% respect to untreated cells. For the other R the luciferase gene knock down after 32 ACS Paragon Plus Environment

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24h was higher than 4h incubation time but do not reach the very high silencing of R5 and R10. Moreover, as demonstrated by transfection results after 48h incubation, it is still detected a knock down effect of nanoaggregates incorporating siRNA at greater R value, i.e. when the INU-IMI-DETA concentration is higher. Transfection experiment by using ICONs containing a scrambled siRNA which cannot target luciferase mRNA instead of siGL3 have been also performed, confirming siGL3 specificity (see Figure S3 in Supporting Information). When transfections were repeated in the presence of Bafilomycin A1 (BAF A1) (Figure 14B), a specific inhibitor of the vacuolar ATPase proton pump found in early endosomes, transfection after 4h for R5 and R10 does not change in terms of effectiveness respect that without treatment with the inhibitor; instead, after 24h a clear difference can be observed, being the transfection activity of the nanoaggregates decreased by a very high extent. The fact that BAF A1 prevent the knock down effect, suggested the involvement of the imidazole mediated proton sponge effect after 24h of incubation in endosomal escape. Starting from R15 a difference of transfection effectiveness with and without treatment with BAF A1 is immediately visible after 4h incubation, suggesting an earlier involvement of the imidazole mediated proton sponge effect respect to R5 and R10. Augmenting the copolymer excess (R15-R35) the difference of transfection in the presence or not of BAF A1, is increasingly higher being concomitantly the copolymer excess much more.


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Figure 14. Luciferase reporter gene down-regulation assay without (A) and with (B) bafilomycin A1 (BAF A1) of ICONs at various INU-IMI-DETA to siGL3 weight ratios (R) and naked siRNA for comparison.

Optimal weight ratios for ICONs effect after 24h incubation was found to correspond to R5-10. At this ratios the overall charge of the particles is slightly negative (Figure 8), a fact which in principle may not be considered optimal due to the negatively charged surface of the cell membrane. However, it was previously observed 31,32,7 that siRNA-carrying liposome/polymeric particles displayed optimal transfection efficacy of siRNAs in different cell types, despite having a negative surface charge, even in cases with a more negative surface charge. Thus, present and past findings suggest that, at least in some tumoral cell types, a mild negativity of the surface of the carrier particle is favourable for siRNA


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delivery. It has also to be considered that the high salt concentration after nanoaggregates formation in DPBS shielded the electric charge of the complexes and that this counterion layer could be replaced/reduced after incubation with other buffer systems and substances present in culture medium. To further understand the impact of complex nanoaggregates composition, and to find clearness in the scenario traced by transfection results, we characterized the cellular uptake and intracellular fate in MCF7 cells by using fluorescently labeled siRNA (siGL3-Cy5). INU-IMI-DETA/siGL3-Cy5 complex nanoaggregates (ICONs-Cy5) uptake studies were conducted following 4 and 24h of incubation and a subsequent extensive washing with DPBS to eliminate out-bounded ICONs-Cy5, by evaluation of recovered fluorescence quantitatively (Figure 15) and also qualitatively (after 4h incubation) by mean of microscope analysis (Figure 16). ICONs-Cy5 were able to be taken up by cells starting from 4h at all the weight ratio tested, with a consistent increase in the uptake at 24h, corresponding to averagely 36.56% of the applied dose per well. Therefore, from these data can be pointed that the uptake does not change significantly upon R variation while an increased uptake occurs as a function of time, going from 4 to 24 h of incubation. Consequently, a greater siRNA uptake cannot be evoked to explain the enhanced gene silencing activity at 4h of incubation for highest R, but further investigations at sub-cellular level were necessary.

Figure 15. Quantitative determination of cellular uptake after 4 and 24h incubation of ICONs/siGL3-Cy5 at INU-IMI-DETA/siGL3 weight ratios of 10, 20 and 30, in MCF-7 cells. Results were compared each


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other and with the siGL3-Cy5 naked uptake results after 24h incubation. (Neither data reported in the graph a) or b) are statistically different when compared each other, p > 0.05).

Figure 16. Qualitative uptake evaluation in MCF-7 cells after 4h incubation of ICONs/siGL3-Cy5 at INU-IMI-DETA/siGL3 weight ratios of: (a) 5; (b) 15; (c) 20; (d) 25; (e)30; (f) 35.

Indeed, the endosomal entrapment of nanoaggregates following the endocytosis was investigated as a critical step in siRNA delivery, as it is believed that the bypass or the escape from the endosomal entrapment (and subsequent lysosomal degradation of siRNA) should be essential for successful RNAi in the cell cytoplasm. Thus, the colocalization of siRNA-Cy5 delivered by ICONs R10 with the late endosome/lysosome was observed by fluorescence microscopy imaging (Figure 17). Experimental condition were chosen considering that R10 is the best performing ratio and that while after 4h incubation


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there is no effect on luciferase expression, after 24h the knock down was over 80%. In this experiment, late endosome/lysosome and the cell nuclei in MCF-7 cells were stained with LysoTracker Geen (shown in green) and DAPI (shown in blue), respectively. After 4h incubation, the colocalization (%) of siRNACy5 with the late endosomes/lysosomes was calculated by dividing the area of colocalized Cy5 signal (yellow) with the total Cy5 signal area (yellow and red). This analysis showed that after 4h of incubation approximately 74% of siRNA-Cy5 in R10, being overlapped with signal of LysoTracker, was entrapped in the endosome/lysosome compartments and that only 25% of Cy5 fluorescence not overlaid the lysotracker signal, that correspond to siRNA not localized into endosome/lysosome compartment. The result is in agreement with transfection of ICONs R10, where after 4h a silencing effect is not yet visible. After 24h, together with the increase of the ICONs amount taken up by cells, the system evades endosomal compartment, thanks to the excess of copolymer that destabilize the endosomal membrane at acidic pH (DETA effect) and mediate the proton-sponge effect (IMI effect), leading to about 80% reduction of luciferase expression. The copolymer mediated endosomal escape is crucial as demonstrated by ICONs R10 transfection in the presence of BAF A1.


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Figure 17. (A) Intracellular distribution after 4h incubation of siRNA-Cy5 delivered by INU-IMI-DETA nanoaggregates (R10) in MCF-7 cells stained with LysoTracker Green and DAPI for the late endosome/lysosome and the nuclei, respectively. (B) Colocalization of siRNA-Cy5 (R) and LysoTracker Green (G).

Conclusions In the present study, the pH-responsive INU-IMI-DETA polycation, obtained through inulin functionalization with imidazole and diethylenetriamine molecules, was developed for enhanced siRNA carrier preparation by the complexation/precipitation method, obtaining Inulin COmplex Nanoaggregates (ICONs). High siRNA encapsulation was achieved even at lower polymer to siRNA weight ratio. ICONs are promising candidates for therapeutic siRNA delivery, because of their ability to overcome specific cellular and subcellular barriers. In fact, upon uptake into endosomal compartments, ICONs are capable of endosomal escaping by disrupting the endosomal membrane thanks to 1,2-diaminoethane destabilising effect and imidazole promoted proton sponge effect. All discussed results support strongly that ICONs evades efficiently the endosomal/lysosomal entrapment, and that the time of appearance and duration of the knock down effect resulted strictly influenced by the weight excess of copolymer employed in ICONs formation; at low R (R10) a fast and strong 24h effect (≈ 80% silencing) is achieved, while when the excess of copolymer employed in nanoaggregates formation is higher (R30), a rapid escape (silencing effect is visible yet at 4h) and translocation to the cytoplasm toward the enhanced and prolonged up to 48h gene silencing activity is reached. Considering that functionalizable groups are still present on the structure of INU-IMI-DETA, the attachment of specific ligands to polymeric carrier or ICON surface, a strategy that is understood as active targeting, could be the next design strategy to increase the uptake in a target cell population and to decrease the uptake in off-target cells and organs. With this approach, the dose administered could potentially be decreased, and the uptake in organs such as the liver and spleen that mainly take up nanoparticles is supposed to be avoided. 38 ACS Paragon Plus Environment

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Supporting Information In vitro haemo-compatibility of INU-IMI-DETA compared with INU-IMI and INU-DETA, in vitro cytocompatibility of INU-IMI-DETA and ICONs on MCF-7 cells, transfection experiments performed by using ICONs (R5-R35) containing scrambled siRNA instead of siGL3, list of abbreviations, representative dimensional distribution of ICONs (R20) measured by AFM.

*Corresponding author. Tel.: +39 91 23891931; fax: +39 91 6100627. E-mail address: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The authors thank the Italian Minister of Instruction, University and Research (MIUR), PRIN 2010-11, [20109PLMH2] for fundings.

Acknowledgment The authors would like to thank Giulia Di Prima, PhD Student of the Department of Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF) of the University of Palermo for her support on 1H NMR characterizations.


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Table of Contents Graphic


Improvements in Rational Design Strategies of Inulin Derivative

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