phosphate supramolecular nanocarriers for intracellular siRNA delivery

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Exploring the pH sensitivity of poly(allylamine) phosphate supramolecular nanocarriers for intracellular siRNA delivery Patrizia Andreozzi, Eleftheria Diamanti, Karen Rapp Py-Daniel, Paolin Rocio Cáceres-Vélez, Chiara Martinelli, Nikolaos Politakos, Ane Escobar, Marco Muzi Falconi, Ricardo Bentes Azevedo, and Sergio Moya ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11132 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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ACS Applied Materials & Interfaces

Exploring the pH sensitivity of poly(allylamine) phosphate supramolecular nanocarriers for intracellular siRNA delivery Patrizia Andreozzi a*, Eleftheria Diamanti a, Karen Rapp Py-Daniel b, Paolin Rocio CáceresVélez b, Chiara Martinelli c, Nikolaos Politakos a, Ane Escobar a, Marco Muzi-Falconi c, Ricardo Azevedo b, Sergio E. Moya a*. a

Soft Matter Nanotechnology Group, CIC biomaGUNE, Paseo Miramón 182 C, 20014 San

Sebastián, Guipúzcoa, Spain. b

Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Genética e

Morfologia, Brasília, Distrito Federal, Brazil. c

Department of Biosciences, Via Giovanni Celoria, 26, University of Milan, 20133 Milan, Italy.

KEYWORDS: self assembly, supramolecular nanocarriers, pH sensitive, drug delivery, siRNA.

ABSTRACT

Silencing RNA (siRNA) technologies emerge as a promising therapeutic tool for the treatment of multiple diseases. An ideal nanocarrier (NC) for siRNAs should be stable at physiological pH

ACS Paragon Plus Environment

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and release siRNAs in acidic endosomal pH, fulfilling siRNA delivery only inside cells. Here, we show a novel application of polyamine phosphate NCs (PANs) based on their capacity to load negatively charged nucleic acids and their pH stability. PANs are fabricated by complexation of phosphate anions from phosphate buffer solution (PB) with the amine groups of poly(allylamine) hydrochloride as carriers for siRNAs. PANs are stable in a narrow pH interval; from 7 to 9, and disassembly at pHs higher than 9 and lower than 6. siRNAs are encapsulated by complexation with poly(allylamine) hydrochloride before or after PAN formation. PANs with encapsulated siRNAs are stable in cell media. Once internalized in cells following endocytic pathways PANs disassembly at the low endosomal pH and release the siRNAs into the cytoplasm. Confocal Laser Scanning Microscopy (CLSM) images of

Rhodamine Green

labelled PANs (RG-PANs) with encapsulated Cy3-labelled siRNA in A 549 cells show that siRNAs are released from the PANs. Co-localization experiments with labeled endosomes and either labelled siRNAs prove the translocation of siRNAs into the cytosol. As a proof of concept, it is shown that PANs with encapsulated green fluorescence protein (GFP) siRNAs silence GFP in A549 cells expressing this protein. Silencing efficacy was evaluated by flow cytometry, CLSM and western blot assays. These results open the way for the use of poly(allylamine) phosphate nanocarriers for the intracellular delivery of genetic materials.

INTRODUCTION Nanocarriers (NCs) provide a means to encapsulate drugs that are not suitable for direct delivery and can be vectorized to reach specific organs and cell compartments for targeted delivery.

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Several strategies have been designed for the encapsulation of drugs using NCs. Encapsulation can take place in the NC core or the drug (therapeutic) can be linked to the NC surface,


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covalently, by electrostatics or through hydrogen bonding interactions depending on the nature and characteristics of both the drug and the NC surface.

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For large therapeutics, e.g. siRNAs,

plasmids, antibodies, their encapsulation in the core of polymeric NCs has the drawback that the release of the encapsulated molecule is limited by the molecule size, usually requiring the degradation of the carrier, which can be slow and reduce the efficacy of the delivery. 8-10 A way to encapsulate large therapeutics is to assemble them on top of NCs by means of the Layer by Layer (LbL) technique as shown for siRNAs

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or for antibodies like antiTNF-α.

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The optimal

situation for nucleic acid delivery would be that the nucleic acid remains encapsulated during NCs circulation and is released into the cellular cytosol after their cellular uptake. 13, 14 In recent years, RNA interference or silencing RNA (siRNA) technologies have emerged as a promising therapeutic tool for the treatment of different diseases, e.g. cancer. 15-20 siRNAs can be applied to treat many cancer types as they can inhibit the expression of multiple genes of interest, being highly specific to a cancer type. 21 However, a major hurdle for their clinical translation is to find a suitable and safe delivery vehicle for the siRNAs.

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Unmodified siRNAs are unstable in

bloodstream due to nuclease degradation, and they can also be immunogenic. 24 Besides, siRNAs are not able to cross cell membranes.

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In the past decade, a variety of NCs designed upon

polymers, cationic lipids, and liposomes have been applied to overcome these limitations.

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An ideal NC for siRNAs delivery should be stable at neutral pH and release siRNA only in the cytoplasm. The development of NCs that are stable during circulation and only release their cargo once inside the cell is indeed of high interest.

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NCs based on the self assembly of

polyamines and phosphate ions are an interesting system for drug delivery.

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These NCs are

very easy to prepare requiring just the mixing in a proper molar ratio of polyamines and phosphate ions, usually from sodium phosphate dibasic solution (Na2HPO4). Polyamines are


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cationic polymers and generate an osmotic difference inside endosomes as they act as a “proton sponge” complexing with protons present in the acid endosomal environment and swelling. This swelling and the osmotic difference created can lead to endosome rupture. The endosomal rupture can be used to transfer a cargo complexed to the polyamines into the cytoplasm.

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Polyamine-phosphate NCs have been used, for example, to encapsulate indocyanine green (ICG) in order to avoid rapid clearance and nonspecific vascular plasma binding, which decreases ICG performance.

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However, no attends have been performed to encapsulate large molecules like

nucleic acids, i.e. siRNAs in the polyamine phosphate NCs. Moreover, the stability of the polyamine phosphate NCs against pH has not been explored for triggering endosomal delivery. We will show that polyamine phosphate NCs (PANs) are stable in a narrow pH interval from 7 to 9, and that they disassembly at pH higher than 9 and lower than 6. This dependence with pH can be used to load PANs by adding water soluble drugs at high or low pH where the polyamines remain as free molecules in solution and then decrease or raise the pH to neutral values to induce NC formation. PANs will trap molecules present in the solution. Alternatively, if the molecule of interest is negatively charged it can form a complex with the polyamines and then by addition of phosphate salts NCs will be formed. Molecules could also be complexed to the NCs after their formation. We will show that the formation of complexes is particularly suitable for encapsulating and delivering siRNAs. NCs with encapsulated siRNAs are stable in cell media without disassembling. Once internalized in cells, following endocytic pathways, the NCs will not be any longer stable and disassembly at the low endosomal pH releasing the siRNA into the cytosol. The efficacy of this approach will be shown for the silencing of the Green Fluorescence Protein (GFP) protein in the GFP-A549 cell line. To resume, we propose here a novel application of poly(allylamine) phosphate NCs for encapsulation and intracellular delivery of siRNAs based


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on their unique pH responsiveness, being stable in cell media and releasing their cargo intracellularly at low endosomal pH. RESULTS AND DISCUSSION Nanocarriers synthesis and pH responsiveness: PANs are prepared in a one step synthesis by mixing PAH and PB as described in materials and methods. Polyamine phosphate NCs are built on the basis of electrostatic and hydrogen bonding interactions between the phosphate ions and the primary amines of the polymers.

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. Besides being a source of phosphate ions, the PB

solution buffers the pH to neutral values, which are necessary for the PANs formation. The size of PANs can be modulated by changing the molar ratio of PAH and PB. In Figure 1 it can be observed that the size of the PANs increases with PB concentration, as measured by Dynamic Light Scattering (DLS). PANs were prepared by adding between 1 to 20 µL of a 10-100 mg/mL PAH solution to 1 mL of a 0.1-10 mM PB to get a final concentration of polymer in the range 0.01-2 mg/mL and in the presence of different concentrations of NaCl, from 0 to150 mM. After mixing the PAH solution with PB in a molar ratio between phosphate and amines (R= PB/NH2) > 1 and a concentration of PAH higher than 0.1 mg/mL, the solution immediately becomes turbid, hinting PANs formation.35. The onset of the PANs formation was determined by DLS, measuring the increase of the scattered intensity expressed as kilocounts (kcounts). We found a ratio of phosphate and amines below which NCs formation is not detectable by DLS, due to the low scattering intensities comparable to the scattered intensity of PAH free-polymer in water. In Figure 2A, the kcounts were plotted against PB concentration for a constant amount of PAH and constant NaCl concentration (10 mM). Kcounts increased as the amount of PB increased, up to a constant value. The maximum inflection of the curve is observed at 5 mM PB, indicating the minimal molar ratio of PB and PAH, i.e. = ] × + ] × 2 ⁄] ×


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~ 1.2,

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necessary for PANs formation that can be detected by DLS. A more detailed

characterization by DLS and TEM was performed for PANs prepared by mixing 10 µL of 100 mg/ml PAH (6.7 x 10-5 M, 15000 MW) in 1 mL of 5 mM PB solution in 10 mM of NaCl, Figure 2B. The hydrodynamic diameter of PANs determined from the intensity distribution was 80 + 25 nm. Similar results were obtained by CUMULANT analysis, where the size of PANs was 66 nm with a polydispersity index (PdI) of 0.2. TEM micrographs corroborate these results as shown in Figure 2C. The average size of the PANs from TEM measurements is 65 + 15 nm, as calculated from the statistical distribution in, Figure 2D. In presence of an excess of phosphate groups R > 4, PANs tend to aggregate. This was interpreted as result of Ostwald ripening by Murthy et al. 35 Conversely, when the molar ratio of PB/NH2 is < 4, PANs are stable in size over a long period of time (17 hours at 25 °C), as shown in Figure S1A and exhibit the lowest PdI, Figure S1B. Moreover, the size of PANs depends on the ionic strength of the solution, Figure 3. The increase in kcounts associated with PANs formation vs the concentration of PB is reported for ionic strengths from 0 up to 150 mM NaCl, Figure 3A. The derived light scattering intensity (kcounts) at 5 mM PB increased with increasing NaCl concentration. This corresponds to an increase in size of PANs from ~60 nm in aqueous solution up to 250 nm at 150 mM NaCl, Figure 3B. DLS measurements were also performed to study the stability of PANs against a wide range of pH, 3 < pH < 12, by adding a few µL of 1 M HCl (pH < 7) or 1 M NaOH (pH > 7). We observed no variations in the scattered intensity of PANs at pH between 7 – 9, Figure 4A. At acidic pH, < 6, the scattering intensity decreased to ~20 kcounts, a value very similar to the scattering of an aqueous PAH solution (free PAH). The decrease in the scattering is a consequence of the PAN disassembling as the phosphate groups lose their charge in an acidic pH. Similarly, the disassembly process is observed when the pH is raised over 9, where the amines of PAH are


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deprotonated, obtaining a low value of scattered intensity. However, when the pH is set again to values between 7 and 9 from either acidic or basic conditions the scattering intensity increased again to the original scattering values before the disassembly of the PANs, resulting an opalescent solution (video 1, SI). The process was monitored in a continuous way measuring the samples by short runs of 10 sec until the kcounts reached a constant value, Figure 4B. These experiments show that the assembly of PANs is a reversible

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processes, meaning that we can

“open” and “close” PANs by tuning the pH, Figure S2A. After disassembly and re-assembly, PANs retain the same characteristics of the original PANs in terms of size and stability, Figure S2B. AFM measurements in dry state of the PANs reveal nanoparticles with an approximately spherical morphology and a height of around 60 nm, Figure 5A. We observe flat polymer structures with heights below 5 nm, Figure 5B, when PANs were deposited on a silica surface, exposed to 0.5-2 µL of 1 M HCl and dried. The polymer from the PANs remains to a large extend at the place where the PANs were located since they were electrostatically attached to the silica surface forming areas of dense polymer content. AFM imaging of pure PAH deposited on silica from drop casting reveals similar height as in the PANs treated with HCl but the coverage of the surface is more homogenous as the polymer deposit from solution, Figure 5C. The AFM images corroborate the results from DLS and the range of pHs at which PANs are stable. Preparation and Characterization of PANs loaded with siRNA. We have followed three different approaches for the loading of siRNAs in the PANs, as sketched in Figure 6. The complex formation and the loading procedures were characterized by DLS and ζ-potential, as reported in Table 1. Procedure 1: post-addition of siRNA. PANs were synthesized starting from 1 mg/mL PAH in 5 mM PB with 50 mM NaCl. To study the interaction with siRNA, PANs were


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diluted up to 0.1 mg/mL with a final concentration of 10 mM NaCl. The hydrodynamic diameter of PANs was 116 nm, with a PdI of 0.04 measured by DLS. Then, 10 µL of siRNA were added to the solution and left to equilibrate for 10 minutes at room temperature. After the addition of siRNA, the PdI increased from 0.04 up to 0.16, conversely the size of PANs after the siRNA complexation remained constant. The siRNA adsorption was confirmed by ζ-potential measurements. ζ-potential values decreased from +47 (PANs) up to +31 mV after the addition of siRNA (PANs + siRNA). This indicates that the positive charges of the amines are partially compensated by the negatively charged siRNA confirming that the plasmid is attached onto the PANs surface. Procedure 2: pre-formation of the PAH + siRNA complexes. Briefly, 10 µL of siRNA were added to 0.1 mg/mL of PAH previously dissolved in RNAse free H2O, and left to equilibrate for 10 minutes at room temperature. The complexes made of PAH + siRNA were formed by the interaction of the amino groups of PAH with the phosphate groups of siRNA. The complexes were characterized by DLS and ζ-potential. Measurements showed that kcounts increased as the amount of siRNA increased, Figure S3. The size and PdI of these complexes are larger compared to the siRNA adsorbed onto the PANs surface. This is probably due to the excess of PAH compared to the amount of siRNA. PAH is added in excess, as the complexes must have an excess of amine groups for the formation of the NCs in presence of phosphate ions. The ζpotential of these complexes is very similar to the ζ-potential of naked-PANs, around +47 mV. Finally, when 1 mM PB was added to the solution PANs with encapsulated siRNA was produced. The final NCs have similar size and ζ-potential of procedure 1, data not shown.


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Procedure 3: Encapsulation of siRNA during PANs formation. 10 µL of siRNA were added to 1 mM PB in 10 mM of NaCl. Then, 0.1 mg/mL PAH (R= PB/NH2 ~ 1.7) was added to this solution. PANs are formed when PAH is added to the PB solution in presence of siRNA. Both PAH/siRNA complexes and NCs are formed simultaneously. As a control PANs with a ratio of PB/NH2 ~ 2 were prepared. From DLS data PANs without entrapped siRNA show an hydrodynamic diameter of 42 nm and a PdI of 0.34. When PANs are formed in presence of siRNA the diameter increases to 83 nm with a PdI of 0.16, indicating the complete entrapment of siRNA into the PANs. Indeed, as reported in the Table 1 the size and ζ-potential of PANs obtained by Procedure 3 are similar to the observed for Procedure 1. The TEM images shown in Figure 7 show that the average sizes of PANs loaded with siRNA following the three different procedures reported above are in good agreement with DLS data.

Cell Entry Mechanism and Co-localization. Before performing the cellular uptake and siRNA transfection experiments, the cell viability was evaluated for the A549 cell line up to 72 h using PANs in a concentration range of 10-3-10-1 mg/mL, which corresponds to 0,01 − 1.1 mM when expressed in terms of PAH monomers (Mw PAH monomers: 94). At all time points a good cell viability (≥ 80%) was observed for PANs up to concentrations of 0.1 mM. This concentration is above the polymer concentration used in the following cell studies in this work. Over 48 h of incubation, a 50% of reduction of the metabolic activity was found when using concentrations ≥ 0.16 mM, Figure 8. Cell viability for PANs is in agreement with the values observed for cationic polymers used for gene delivery, i.e. polyethyleneimine (PEI), a frequently used polycation in transfection, has been shown to induce a lower cell viability than PANs for the same concentration range.45,46 In light of these results, all cellular experiments were performed using a


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final PANs concentration of 5 x 10-3 mg/mL (5 x 10-5 M) for long term incubation (> 24 hours) and 1 x10-2 mg/ml (1 x 10-4 M) in case of short time incubation (< 3 hours). Cellular internalization of fluorescent Rhodamine labelled PANs (RB-PANs) was quantified via flow cytometry. The fluorescence intensity, corresponding to the maximum uptake efficiency was found for a PANs concentration of 0.01 mg/mL after 2 hours of incubation at 37 °C, Figure S4. In order to confirm the co-localization of the PANs in the endosomal compartments of the cells, experiments with different endocytic pathways for the A549 cell line were performed. Early Endosome Antigen-1 (EEA-1) and Transferrin receptor 2 (TFR2) were used as markers for the early endosomes, while the Lysosomal Associated Membrane Protein -1 (LAMP-1) was used to visualize the lysosomes. Co-localization experiments were performed by CLSM. PANs are distributed in the cells in endocytic vesicles, suggesting that endocytosis is the entry mechanism followed by the PANs. In particular, PANs entered the A549 cells and accumulated in the early endosomes and lysosomes, as deduced by the co-localization of the red fluorescence of PANs, and the green fluorescence of EEA-1 and LAMP-1 antibody, Figure 9. Increased co-localization of RB-PANs was observed after 2 h of post-incubation. Similar co-localization was found with TFR2, marked recycling endosomes, Figure S5. Intracellular Trafficking and Escape of siRNA in vitro. It is known that the successful escape from endosomes is crucial for siRNA nanocarriers to improve siRNA silencing efficiency. Moreover, for silencing the siRNA must be released from the PANs. The intracellular fate of siRNAs was studied in A549 cells by means of CLSM. For these experiments, Cy3-labelled siRNAs were encapsulated in Rhodamine green labelled PANs (RG-PANs). PANs were prepared with Rhodamine green labelled PAH polymer (RG-PAH). Rhodamine green and Cy3 have non overlapping fluorescence spectra, which allow us to trace the fate of the PANs and the


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siRNAs separately. RG-PANs carrying Cy3-labelled siRNA display a yellow color in the confocal microscope as consequence of the superposition of the green color of the PAH and the red of the siRNAs, while green spots correspond to PANs without siRNAs and red spots to the free siRNA. These images give an additional proof of the siRNA encapsulation in the PANs. Cells were transfected with Cy3-labelled siRNA/RG-PANs prepared following the Procedure 1, using as transfection medium PB solution, with a final concentration of 5 x 10-3 mg/mL in cell media. The images were acquired after 4 h of incubation, Figure 10. A high degree of colocalization (yellow signal) from RG-PANs (green) and Cy3-labelled siRNA (red) was observed inside the cell, indicating that PANs are delivering siRNA intracellularly. In addition, red spots can be observed in the cytosol resulting from siRNA that do not co localize with the RG-PANs. These correspond to the siRNAs released from the PANs. Co localization results are confirmed by a z-stack acquisition (video 2, SI). CLSM data proves the opening of the PANs and the delivery of the siRNAs intracellularly. To determine if the siRNA escape from the endosome cells were transfected with PANs (not labelled) loaded with Cy3-labelled siRNA, and endosome/lysosomes were then stained with LysoTracker Green at 2 h post-transfection. LysoTracker Green is a fluorescent acidotropic probe for labeling and tracking acidic organelles in live cells, such as endosomes and lysosomes. Cy3-labelled siRNA/PANs reached the endosomes/lysosome after 4 h of incubation, as is shown from the co-localization of the red signal of Cy3-labelled siRNA and green fluorescence signal of LysoTracker in Figure 11. Non encapsulated Cy3-labelled siRNA and Cy3-labelled siRNA/Lipofectamine complexes were used as controls. While the free Cy3-labelled siRNA is not found in the cells we observe for both the lipofectamine and the PANs with Cy3-labeleld siRNA a similar intracellular fate. In both cases siRNA signals co-localize with LysoTracker Green as observed from the yellow clusters


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resulting from the superposition of the red signal of the siRNAs and the green from the endosomes. In addition, red clusters of siRNAs that are not co-localized with Lysotracker Green are observed, meaning that siRNAs follow endosomal escape.

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Co-localization experiments

bring significant insight in the fate of the siRNAs and PANs proving in the case of the dual labelling of PANs and siRNA that the PANs deliver the siRNA intracellularly and that the siRNAs are released, which is expected as the PANs should open inside the endosomes. The colocalization experiments with endosomes prove the translocation of the siRNAs into the cytosol, which is necessary for the silencing. Furthermore, detailed DLS analyses were conducted to evaluate the stability and the release of siRNA-PANs at a pH similar to the endocytic compartment. As inferred by DLS, the siRNA-PANs prepared by the three different loading procedures dissemble into molecular components after being treated with 0.5 M HCl to get a pH < 5, Figure S6. Effectively, PANs loaded with siRNA are unstable at pH < 5 obtaining similar kcounts as for free PAH. TM replacing PB was used as a control in the transfection experiments. The hydrodynamic diameter of PANs loaded with siRNA following the three different loading procedures in TM, Figure S7 was similar to the obtained adding PB to the same preparation procedures reported in Table 1. siRNA GFP delivering. We used PANs for gene delivery due to their capacity to spontaneously disassembly in the pH range of endosomes and the capacity of the free poly(allyamine) to act as membrane-disruptive,

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releasing the genetic materials into the cytoplasm. The GFP-A549

cell line was used as a model in the silencing experiments. This cell line was chosen because it has a stable expression of the GFP protein that can be knocked down by the delivery of siRNAs. A flow cytometry analysis was carried out to quantify the efficacy of siRNA delivered by PANs with Lipofectamine as control. Flow cytometry allows to evaluate the efficacy of the GFP


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silencing measuring the fluorescence intensity per cells after transfection experiments. The transfections were performed using a final concentration of PANs in contact with the cells of 5 x 10-3 mg/mL. Cells were transfected with siRNA-PANs prepared with the 3 different procedures described in the material and methods, using as transfection reagent PB solution or alternatively, TM. The efficacy of the siRNA-PANs was compared with siRNA delivered by Lipofectamine, which is known to effectively silence GFP. The expression level of GFP in the GFP-A549 cells, in terms of fluorescence intensity, decreases by 89% compared to untreated cells when these are transfected with Lipofectamine, Figure 12. The decrease of the fluorescence intensity of cells transfected with siRNA-PANs prepared by the three different procedures corresponds to an inhibition of GFP expression of approximately 60-65%. When naked-siRNA was used as a control, no GFP inhibition was observed. Similar results were obtained with the samples prepared in transfection medium (TM), Figure S8. CLSM images confirm the silencing efficacy of PANs. CLSM fluorescence images were acquired to visualize the GFP fluorescence in the GFP-A549 cells before and after siRNA delivery. In parallel cells were imaged in transmission mode as mean to quantify the number of silenced cells and evaluate the efficacy of the silencing. In Figure 13, transmission images, fluorescence images and the merging of both images are shown. GFP-A549 cells show an intense green fluorescence. 100 % of the cells counted show fluorescence and practically no differences in fluorescence intensity can be appreciated among cells, Figure 13a. The delivery of the naked siRNA does not affect the fluorescence intensity, however, the number of cells expressing GFP is reduced to an 87 %, Figure 13b. The silencing with the siRNA PANs in Figure 13c results in a 27 % the counted cells displaying fluorescence. Cells transfected with siRNA/PANs expressing GFP show a lower fluorescence intensity than the control. The delivery


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of siRNA as Lipofectamine complexes results in the silencing of most of the cells. Only an 8 % of the counted cells still show fluorescence, Figure 13d. As for the PANs, cells expressing GFP after lipofectamine delivery show a lower fluorescence intensity than the control. CLSM images corroborate the trend shown by the flow cytometry. The siRNA PANs are effectively silencing the expression of GFP in GFP-A549 cells but are less effective than Lipofectamine complexes. In addition, we have performed a western blot determination of GFP in GFP-A549 cells after siRNA/PANs transfection. Samples were prepared with the three different procedures described before. The experiment confirms a decrease in the GFP signal after the transfection, even though in a lesser degree than for the siRNA delivery with Lipofectamine, Figure 14. It is worth mentioning that the intensity of the band of the GFP signal for the Lipofectamine is almost 0.40.3 of the value observed for the not treated cells as reported in the quantification lane in Figure 14. In case of samples transfected with siRNA/PANs using the three different procedures the decreasing of GFP band was less significant, in the best case to 0.7. Better results were obtained for the samples prepared in transfection medium with a decrease of 0.6 for the PANs, while a 0is observed for the lipofectamine. It is worth mentioning that in general the decrease in GFP expression determined by western blot is comparatively not as significant as the decrease in fluorescence observed with flow cytometry or from CLSM images. This could be due to the fact that A549 cells constitutively overexpress GFP, a very stable protein when expressed intracellularly. Overall, the silencing of siRNA-PANs, being significantly more effective than naked siRNA, hints an efficient association between the siRNAs and the PANs for siRNA delivery. Despite that siRNA delivery by Lipofectamine resulted in a higher decrease in fluorescence intensity, the


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delivery by PANs effectively silenced the GPF up to almost a 70 %, which is a highly acceptable values for silencing and encourage the use of PANs for siRNA delivery. Conclusions. Nanocarriers with tunable size are fabricated by simply mixing PAH and PB in different molar ratios. A balance of charges between PAH and PB, defined by their pK, determines PANs stability with pH. At pH > 9 the deprotonation of the amines in PAH results in the disassembling of the carriers. Similarly, at pH < 6 the phosphate groups lose their charges and the PANs disassemble. Our results show that the carriers are stable only in a range of 6 < pH < 9 and that they can be reversibly assembled and disassembled by changing pHs to values in and out of their stability window. We have encapsulated siRNAs in the PANs using three different procedures. We observe that the GFP siRNAs are released inside GPF-A549 cells as they effectively silence the GPF to an extent comparable with Lipofectamine delivery of the same siRNAs. PANs carrying siRNAs are stable in cell media, pH 7, and they release the cargo in endosomes, where the pH is 5 and the carriers are not stable. The nanocarriers proposed here are an interesting alternative to Lipofectamine as they are easily prepared, based on inexpensive components and disassemble at pH conditions that can be only found in the endosomes. The same approach could be used to deliver other genetic materials. Further work on the functionalization of the NCs for specific targeting is ongoing.


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Experimental Section Materials. Poly(allylamine) hydrochloride salt (PAH) (MW: 15 x104 and 70 x104 g/mol), Phosphate Buffer Salt tablets (PBS), Sodium Phosphate dibasic (Na2HPO4), Potassium Phosphate monobasic (KH2PO4), Hydrochloric Acid (HCl), Sodium Hydroxide (NaOH) and Sodium Chloride (NaCl), all from Sigma-Aldrich, were used as received. Polyelectrolyte stock solutions and all subsequent diluted precursor solutions were prepared with MilliQ deionized water. Human lung adenocarcinoma (A549 CCL-185) cell lines were purchased from the American Type Culture Collection (ATCC, USA). GFP-A549 cell lines were purchased from Amsbio. Roswell Park Memorial Institute, RPMI medium was purchased from Lonza (USA). Ham’s F-12K medium, fetal bovine serum (FBS Hyclone) silencer GFP (eGFP) RNA, silencer Cy3-labeled GAPDH siRNA, LysoTracker Green DND-26, Lipofectamine RNAiMAX (TR) reagent and UltraPure™ DNase/RNase-Free Distilled Water and Blasticidin were purchased from ThermoFisher. 3,4,5-dimethylthiazol-2,5 biphenyl tetrazolium bromide (MTT), Rhodamine B, Penicillin-streptomycin, Saponin, Goat Serum, Camptothecyn and (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) HEPES were purchased from Sigma Aldrich (USA). Anti-EEA-1 antibody ab2900; anti-LAMP1 antibody, [H4A3] ab25630; Goat Anti-rabbit IgG H&L (Alexa Fluor 488), ab 150077; Goat anti-mouse IgG H&L (Alexa Fluor 488) preadsorbed, ab150117 were purchased from Abcam. Transferrin TFR2 (H-140), sc-48747, and siRNA Transfection Medium (TM), sc-36868 from Santa Cruz Biotechnology, INC. Grids and supports of copper and osmium tetroxide were obtained from Electron Microscopy Sciences (USA). Dynamic Light Scattering (DLS). Dynamic Light Scattering measurements were carried out with a ζ-Sizer Malvern Instrument in backscattering mode. All studies were performed at a 173° scattering angle with temperature controlled at 25 °C in 1 mL polystyrene cuvettes. PANs were


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characterized in terms of size and ζ-potential. Short time measurements were carried out for a total of 15 min, with 3 consecutive measurements for each sample. ζ-potential measurements were performed in auto-mode at 25 °C, with 3 consecutive measurements for each sample. Transmission Electron Microscopy (TEM). For transmission electron microscopy analysis of PANs normal and ultra-thin plasma coated carbon film were used. 2 µL of undiluted PANs with PAH concentration of 0.01-2 mg/mL (assembled 30 min prior to grid deposition of samples) were transferred to plasma coated grids and incubated for 1 min, followed by washing with degassed Nanopure water, incubation with 3 µL of ammonium molybdate 20 mg/mL for 1 min and three final washed with degassed Nanopure water. Transmission electron microscopy analysis was performed by using a JEOL JEM 1010 microscope operating at an acceleration voltage of 100 kV. Atomic Force Microscopy (AFM) imaging. AFM was performed with a Veeco Multimode atomic force microscope attached to a Nanoscope V controller for the morphological characterization of the PANs. The tips for the AFM were purchased from Bruker, TESP-V2 model. All samples were drop casted over a clean Si surface (Si surfaces were previously cleaned at least for 20 min on a UV/O3 oven) and left to evaporate for at least 24 hours before the measurement in dry state. Cell Culture. Human lung adenocarcinoma cell (A549) cell lines were cultured with RPMI 1640 medium supplemented with 10 % (v:v) fetal bovine serum (FBS) and 1 % (v:v) antibiotic solution (100 units/mL penicillin, 100 mg/mL streptomycin, P/S). Cells were maintained at 37 °C, 5% CO2 in humidified chamber. GFP-A549 cell lines were cultured with Ham’s F-12K medium supplemented with 10 % of FBS, 1 % P/S and Blastidicin at 10 µg/mL.


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Cell viability MTT assay. Cell mitochondrial activity was tested using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which is based on the mitochondrial conversion of the tetrazolium salt into a formazan dye with absorption characteristics in the visible region. PANs were incubated with cells at different concentrations and different time points (0 – 72 hours). Following incubation with PANs at each time point, cells were washed and 135 µL fresh medium with 15 µL of MTT (at 5 mg/mL in PBS) were added to each well. Culture plates were then incubated at 37 °C. After 2 hours of incubation, medium-containing MTT was discarded and formazan crystals were dissolved in 150 µL DMSO. The absorbance at 550 nm (with automatic discount of ref wavelength 630 nm) of the resulting solution was measured in a 96-well spectrophotometer microplate reader. Percentage cell mitochondrial activity was determined by the following formula: (Absorbance of treated cells/ Absorbance of control cells) x 100%. Cell Entry Mechanism and Intracellular Trafficking of RB-PANs. The cellular uptake of rhodamine B labelled PANs (RB-PANs) was quantified via flow cytometer. PAH labeled with red (RB-PAH) and Rhodamine green (RG-PAH) were provide by Surflay AC, Germany. Briefly, A549 cells were cultured on 24 well plates and exposed to 0.01 mg/mL of RB-PANs for different time points, from 10 up to 180 min at 5 % CO2 and 37 °C. After PANs incubation the cells were washed with PBS and trypsinized. Fluorescence of the cells was quantified and analyzed with a BD-FACs Canto II cytometer, (Becton Dickinson, USA). Measurements were performed in duplicate and approximately 104 events (cells)/sample were analyzed. A549 cells not exposed to PANs were used as a control. A549 cell incubation with fluorescent labelled PANs (RB-PANs) was performed to evaluate the entry mechanism and the endosomal distribution of PANs at different time points. 4.5 x 104 cells were plated the day before co-


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localization studies in a 24 well glass bottom plate with 500 µL of RPMI full medium. Prior to PANs addition cells were left equilibrate for 45 min at 4 °C in order to block and then synchronize endocytic processes. PANs were added to the cells in RPMI full medium, and allowed to bind to the cell for 1 h at 4 °C. Still on ice, cells were washed with ice-cold PBS to remove unbound PANs. Then, temperature was raised to 37 °C, 5 % of CO2, to start the synchronized uptake, for different time points from 0 up to 120 minutes. Afterwards, cells were washed and indirect immunofluorescence assays with labelled antibody were performed. Early endosome (EEA-1), transferrin (TFR2) as a marker of clathrin-dependent endocytosis and Lysosome (LAMP-1) antibody were used.48,49 Cells were fixed for 10 min at room temperature with (p-formaldehyde) PAF at 4 %, permeabilized with 0.5 % saponin in PBS and then, were treated with blocking solution containing 5 % of goat serum in PBS for 1 hour. Then, cells were left in contact with primary antibodies in a blocking solution, overnight at 4 °C. Cells were incubated with the appropriate fluorescent secondary antibodies in blocking solution, 1 h at room temperature. All the washing steps were performed with PBST++ solution (Tween 20 at 0.05 % in PBS containing 1 mM of Ca2+ and Mg2+). In all cases, cells nuclei were counterstained with DAPI (0.1 µg/mL) diluted in PBS. CLSM imaging was performed exciting at 405, 488, 561 nm excitation laser lines, with 40x and 63x objectives. All the images were acquired by LSM 510 Zeiss laser scanning microscope. Preparation of PANs loaded with siRNA. We have followed three different approaches for the loading of siRNAs in the PANs. Procedure 1: post-addition of siRNA. PANs were synthesized starting from 1 mg/mL PAH (6.7 x 10 -5 M, 15000 MW) in 5 mM PB with 50 mM NaCl, with R = PB/NH2 1.2. Then 2.5 µL of PANs solution were added to an eppendorf with 2 µL of siRNA (~ 0.65 µg, stock solution 50µM). The PANs-siRNA complex was left equilibrate for 15 minutes


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and then 45.5 µL of 1 mM PB in 10 mM NaCl or Transfection Medium (TM) were added to the complex before cell transfection. Procedure 2: pre-formation of the PAH + siRNA complexes. Briefly, 2 µL of siRNA (~ 0.65 µg) were added to the 2.5 µL of 1 mg/mL (2.5 µg) PAH dissolved in a RNAse free H2O, left equilibrate for 15 minutes and then 45.5µL 1 mM PB in 10 mM NaCl or TM were added to the complex. The addition of PB (TM) results in the formation of the PANs. Procedure 3: Encapsulation of siRNA during PANs formation. 2 µL (~ 0.65 µg) of siRNA were added to the 45.5 µL of 1 mM PB in 10 mM NaCl or TM, and then 2.5 µL of 1 mg/mL (2.5 µg) PAH were added to this solution. PANs are formed when PAH is added. In the experiments N/P ratios of PAH/siRNA complexes were the ratios of moles of the amine groups of PAH to those of the phosphate ones of siRNA.50,51 In case of transfection experiments the molar ratio of protonatable polymer amine groups to nucleic acid phosphate groups (N/P) was ~ 2. Intracellular Trafficking and Escape of siRNA in vitro. 4 x 104 A549 cells were plated the day before of the co-localization studies in an 8-well glass bottom plate (Lab-Tek Chambered #1.0 Borosilicate cover glass system, 155411, ThermoFisher) with 400 µL of RPMI at 10 % of serum and 1 % of P/S. Before transfection experiments, cells were washed with medium at 2 % of serum and then, 400 µL of fresh medium containing 2 % of serum without any antibiotic were added to each well. Then 40 µL of Cy3-labelled siRNA/RG-PANs with N/P ~ 2 was added to the cells and incubated for 4 h at 37 °C and 5 % CO2. Naked Cy3-labelled siRNA was used a negative control. Lipofectamine loaded with Cy3-labelled siRNA was used as a positive control for silencing efficacy. Cells were washed twice with fresh medium and imaged by CLSM. To follow the endosomal release of siRNA/PANs, cells were treated with Cy3-labelled siRNA/PANs at 37 C for 2 h. Subsequently, cells were stained with LysoTracker green following


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the manufacturer's instructions. Cells were then washed twice with fresh medium and imaged by CLSM. CLSM imaging was performed exciting at 405, 488, 561 nm excitation laser lines, with 63x objectives. All the images were acquired by LSM 510 Zeiss laser scanning microscope. Transfection experiments. Briefly 2 x 104 GFP-A549 cells were placed in a 24 well, with medium at 10 % of serum and 1 % of P/S the day before transfection. Before transfection experiments, cells were washed with medium at 2 % of serum and then, 500 µL of fresh medium containing 2 % of serum without any antibiotic were added to each well. The transfection was followed for 72 or 96 h. The silencing experiments were performed following the three different procedures reported above. Lipofectamine loaded with siRNA was used as a positive control for silencing efficacy. All the complexes were left equilibrate 15 minutes at room temperature before transfecting the cells. Silencing efficacy was quantified via flow cytometry. Cells were washed with PBS, trypsinized, and fixed in 1 % PFA in PBS for 15 min at room temperature. Approximately 1 x 104 events (cells) were analyzed per sample while untreated GFP-expressing A549 cells were used as a positive control (i.e the fluorescence intensity value of the cells before silencing). The percentage of transfected cells was calculated as the number of GFP+ (positive) cells over the total population of analyzed cells. Experiments were performed with a BD FACSCalibur™ flow cytometer (BD Biosciences) and data were analyzed with the BD CELLQuest™ software (BD Biosciences). In parallel siRNA efficacy was assessed by wield field Inverted Leica microscope, visualizing the decrease in GFP fluorescence as a result of the siRNA delivery. Cells were fixed in 4 % of p-formaldeyde (PFA) in PBS for 10 min at room temperature. GFP-A549 cell were visualized with 40x - 63x objectives. Western blot. Transfected cells were trypsinized 96 hours post-transfection, harvested and lysed in 2X Laemmli sample buffer (125 mM Tris-HCl, pH 6.8, 4 % SDS, 20 % glycerol, 200 mM


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DTT, 0.02 % bromophenol blue), sonicated 10 s and heated at 95 °C for 10 min. Total protein extracts were analyzed by 10 % polyacrylamide gel. TGX Stain freeTM FastCastTM Acrylamide Kit was used to handcast 10 % polyacrylamide gel (Bio-Rad). Protein marker from GeneSpin s.r.l. (StoS Protein Marker) was used. The gel was then transferred on 0.2 µm nitrocellulose membrane using the Trans-Blot® TurboTM Transfer System (Bio-Rad). The following antibodies were used: anti-Actin (Sigma, 1:1000); anti-GFP (Invitrogen, 1:1000). Secondary antibody was goat anti-rabbit conjugated to HRP. Chemiluminescent reaction was performed by using ClarityTM Western ECL Substrate (Bio-Rad). Images were acquired using the ChemiDocTM Touch Imaging System (Bio-Rad) and quantified using Image LabTM Software (Bio-Rad).

FIGURES

Figure 1. Schematic illustration of PANs preparation. In a standard preparation 10 µL of 100 mg/mL PAH are added to the 1 mL of PB solution with/without NaCl. The size of PANs increases as the molar ratio between PB/NH2 increases.


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Figure 2. DLS and TEM characterization of PANs formation. A) Derived light scattered intensity (kcounts) of PAH at 1 mg/mL vs different concentrations of PB in 10 mM NaCl. B) Hydrodynamic Diameter (nm) of PANs made of 1 mg/mL of PAH vs at 5mM of PB in 10 mM of NaCl. C) TEM Image of PANs at 1 mg/mL in 5 mM of PB in 10 mM of NaCl. Scale bar indicate 200 nm. D) Particle size distribution of PANs (Figure 1C) obtained by TEM images counting > 500 NPs. The size average is 65 + 15 nm.


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A

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B

Figure 3. DLS characterization of PANs in presence of different concentrations of NaCl. A) Derived light scattering intensity (kcounts) of PAH at 1 mg/mL vs PB [mM] in different concentrations of NaCl [mM], as reported in the legend. B) Hydrodynamic Diameter (nm) of PANs in 5 mM of PB vs different molar concentration of NaCl [mM].

Figure 4. A) Derived light scattering intensity (kcounts) of PAH at 1 mg/mL in 5 mM of PB in 50 mM of NaCl at different pH. B) The reversible PANs disassembly is reported as derived light scattering intensity (kcounts) of PAH at 1 mg/mL 5 mM of PB in 50 mM of NaCl at different pH


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vs time (sec). The maximum value of kcounts 2-3 x 103 (opalescent solution) are obtained in the pH range between 7-9, indicating the formation of PANs.

Figure 5. 2D AFM height images 500 nm x 500 nm for A) PANs formed from 1 mg/ml PAH in PB 5 mM in 50 mM of NaCl; B) same PANs treated with HCl, at pH around 4 and C) PAH drop casted from a 1 mg/mL aqueous PAH solution. All the samples were drop casted over Si surface.


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Figure 6 Preparation of PANs loaded with siRNA. Procedure 1: post-addition of siRNA. PANs were synthesized starting from 1 mg/mL PAH in 5 mM PB and 50 mM NaCl. siRNAs were added to the PANs solution diluted 1:10 in 10mM NaCl. The complex is left equilibrate and, 1 mM PB in 10 mM NaCl or Transfection Medium (TM) is added before to transfect the cells. Procedure 2: pre-formation of the PAH + siRNA complex. siRNA is added to the PAH dissolved in RNAse free H2O, left equilibrate and then PB 1 mM in 10mM of NaCl or TM are added to the complex. Procedure 3: Encapsulation of siRNA during PANs formation. siRNA is added directly to 1 mM PB or TM solution, then PAH is added to this solution to form PANs with entrapped siRNA.


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Table 1: Hydrodynamic Diameter (nm), Polydispersity Index (PdI), Kcounts and ζ-potential values (mV) of siRNA-PANs prepared with the three different preparation procedures. Measurements were performed at 25 °C. Sample Name

Hydrodynamic Diameter (nm)

PdI

Kcounts

PANs 0.1 mg/mL

116 + 2

0.04 + 0.02

2321 + 107

47 + 1

PANs 0.1 mg/mL+ 10 µL siRNA

126 + 2

0.16 + 0.01

2117 + 5

31 + 2

(PAH 0.1 mg/mL+ 10 µL siRNA) complex

211 + 2

0.40 + 0.01

214 + 3

45 + 4

(PB 1 mM + 10 µL siRNA) + PAH 0.1 mg/mL

83 + 2

0.16 + 0.01

2446 + 327

25 + 1

ζ-potential (mV)

Figure 7. TEM images of siRNA loaded nanocarriers obtained from the three different procedures followed. A) PANs synthesized starting from 1 mg/mL PAH in 5 mM PB with 50 mM NaCl then diluted up to 0.1 mg/mL with a final concentration of 10 mM NaCl. The average size is 80 + 20 nm. B) Procedure 1: post-addition of siRNA. The average size is 110 + 40 nm. C) Procedure 2: pre-formation of the PAH + siRNA complexes without adding PB. The average


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size is 110 + 30 nm. D) Procedure 3: Encapsulation of siRNA during PANs formation. The average size is 90 + 30 nm. Scale bar indicate 200 nm.

Figure 8. % of Cell Viability of A549 cells after exposure at various concentrations of PANs reported as molar concentration of cationic monomers in PAH [mM] and at different set time points from 1 up to 72 hours, as reported in the legend.


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Figure 9. Co-localization studies by Confocal Laser Scanning Microscopy (CLSM). RB-PANs at 0.01 mg/mL were allowed to bind to A549 cells for 60 min at 4 °C; then, the temperature was


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increased to 37 °C for different time periods. Panel (a): immunostaining of EEA-1 antibody at different time points. Panel (b): cells were immunostained with anti-LAMP-1 at different time points. Following 60 min of cellular uptake, PANs are present in EEA-1 positive endosomes (internalization) and LAMP-1 positive lysosomes (degradation). Scale bar: 20 µm.

Figure 10. CLSM images, transmission, fluorescence channels and merged, of Cy3-siRNA/RGPANs cellular uptake in A549 cells. Confocal images demonstrate the cellular uptake of Cy3siRNA/RG-PANs. Images were taken after cells were incubated with NPs for 4 h. Red spots correspond to Cy3- siRNA, while green spots to RG-PANs.. The yellow fluorescence is a result of co-localization of Cy3-labelled siRNA and RG-PANs. The scale bar is 10µm.


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Figure 11. CLSM merged images of A549 cells transfected by naked Cy3-siRNA as a control (a), Cy3-labelled siRNA/Lipofectamine (b), and Cy3-labelled siRNA/PANs (c). The ratio of PANs/siRNA is 10. Images were taken after cells were incubated with NPs for 4 h. The red signal comes from the Cy3-siRNA, while the late endosome and lysosome are stained with LysoTracker Green (green). The yellow fluorescence is a result of co-localization of LysoTracker Green and Cy3-labelled siRNA. The scale bar is 10µm.


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Figure 12. Fluorescence Intensity maximum (a.u.) measured by flow cytometry to evaluate the efficacy of the silencing of GFP expression in the GFP-A549 cell line exposed to PANs carrying GFP siRNAs for the three preparation procedures followed: PANs-siRNA (green bar); (PAH + siRNA) complex (purple bar) and (siRNA+PB) + PAH (blue bar). As control GFP-A549 cells were treated with 1 µL of Lipofectamine loaded with siRNA (red bar). Untreated cells (black bar) and cells treated with naked-siRNA (grey bar) are reported. All samples were treated with the same concentration of siRNA and the same amount of PANs. The error bars represent the standard deviation of duplicate samples.


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Figure 13 CLSM images of not transfected A549 cells (a); cells transfected with 5 µL of naked siRNA (b); cells transfected with 2 µL of siRNA encapsulated after PANs formation at 5 x 10-3 mg/mL (c); and cells transfected with 1 µL of Lipofectamine and 2 µL of siRNA (d). The scale bar indicates 25 µm.


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Figure 14. Western blot analysis of GFP content for A 549 cells after transfection. Samples were analyzed 96 h post transfection. In all the samples the same ratio of N/P = 2, siRNA and the same amount of transfection reagent were used. Actin was used as control. Lane 1: Not transfected cells. Lane 2: GFP expression of cell transfected with siRNA GFP in lipofectamine, Transfection Reagent (TR) in PB. Lane 3: GFP expression of cells transfected with siRNA GFP loaded PANs by Procedure 1 in PB. Lane 4: GFP expression of cell transfected with siRNA GFP loaded PANs by Procedure 2 in PB. Lane 5: GFP expression of cell transfected with siRNA GFP loaded PANs by Procedure 3 in PB. Lane 6: Not transfected cells. Lane 7: GFP expression of cell transfected with siRNA GFP in transfection reagent (TR) in transfection medium (TM). Lane 8: GFP expression of cell transfected with siRNA GFP loaded PANs by Procedure 1 in TM. Lane 9: GFP expression of cell transfected with siRNA GFP loaded PANs by Procedure 2 in TM. Lane 10: GFP expression of cell transfected with siRNA GFP loaded PANs by Procedure 3 in TM. Image quantifications was performed using Image LabTM Software (Bio-Rad).

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AUTHOR INFORMATION Corresponding Author Sergio Moya [email protected] Patrizia Andreozzi [email protected]

Author Contributions P. A. conceived and planned the experiments. P. A. performed dynamic light scattering experiments, co-localization studies, and transfection experiments. P. A., E.D. and A. E. performed co-localization study. P. A., E. D. and C. M. wrote the paper. K. R. P. and P. R. C. V. performed transmission electron microscopy, performed cell viability assay. C. M. preformed the western blot assay. N. P. performed atomic force microscopy, M. M.-F., and R. A. contributed valuable scientific discussion and proof-read the manuscript. S. E. M. supervised the work and wrote the paper. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Project (Project ID: 612453) Viroma Project, (Project ID: 612453), FP7-PEOPLE-2013-IAPP - Marie Curie Action: "Industry-Academia Partnerships and Pathways". Notes: The authors declare no competing financial interest


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ACKNOWLEDGMENT P.A. and S.M. thank the Viroma Project (Project ID: 612453) FP7-PEOPLE-2013-IAPP - Marie Curie Action: "Industry-Academia Partnerships and Pathways". C.M. and M. M.-F. thank Telethon-Italy (grant number GGP15227). ABBREVIATIONS PAH Poly(allylamine) hydrochloride salt; PB Phosphate Buffer; PBS Phosphate Buffer Saline; NCs nanocarriers; PANs Polymeric assembled nanocarriers; TR transfection reagent Lipofectamine; TEM transmission electron microscopy; DLS dynamic light scattering; AFM atomic force microscopy; siRNA silencing RNA; DAPI 4',6-diamidin-2-fenilindolo; EEA-1 Early Endosome Antigen 1; LAMP-1 Lysosomal-Associated Membrane Protein 1; TFR2 Transferrin receptor 2; TM transfection medium.


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phosphate supramolecular nanocarriers for intracellular siRNA delivery

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