Triblock Copolymer Nanovesicles for pH-Responsive Targeted

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Triblock copolymer nanovesicles for pH-responsive targeted delivery and controlled release of siRNA to cancer cells. Elena Gallon, Teresa Matini, Luana Sasso, Giuseppe Mantovani, Ana Armiñan de Benito, Joaquin Sanchis, Paolo Caliceti, Cameron Alexander, Maria Jesus Vicent, and Stefano Salmaso Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00286 • Publication Date (Web): 19 May 2015 Downloaded from http://pubs.acs.org on May 23, 2015

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Triblock copolymer nanovesicles for pH-responsive targeted delivery and controlled release of siRNA to cancer cells. Elena Gallon,a Teresa Matini,b Luana Sasso,b Giuseppe Mantovani,b Ana Armiñan de Benito,c Joaquin Sanchis,c Paolo Caliceti,a Cameron Alexander,a* Maria J. Vicentc*and Stefano Salmasoa* a

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F.

Marzolo 5, 35131, Padova - Italy b

c

School of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.

Centro de Investigation Principe Felipe (CIPF), Polymer Therapeutics Laboratory, Av.

Eduardo Primo Yúfera 3, E-46012, Valencia, Spain

ACS Paragon Plus Environment

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ABSTRACT New pH-responsive polymersomes for active anticancer oligonucleotide delivery were prepared from triblock copolymers. The delivery systems were formed by two terminal hydrophilic blocks, PEG and poly-glycerolmethacrylate (poly-GMA), and a central weakly-basic block, poly-imidazole-hexyl methacrylate (poly-ImHeMA), which can complex with oligonucleotides and control vesicle formation/disassembly via pH variations. Targeted polymersomes were prepared by mixing folate-derivatised and underivatised copolymers. At pH 5, ds-DNA was found to complex with the pH-responsive copolymers at a N/P molar ratio above ~2:1, which assisted the encapsulation of ds-DNA in the polymersomes, while low association was observed at pH 7.4. Cytotoxicity studies performed on folate receptor over-expressing KB and B16-F10 cells and low folate receptor expressing MCF-7 cells showed high tolerance of the polymersomes at up to 3 mg/mL concentration. Studies performed with red blood cells showed that at pH 5.0 the polymersomes have endosomolytic properties. Cytofluorimetric studies showed a 5.5-fold higher up-take of ds-DNA loaded folate-functional polymersomes in KB cells compared to non-targeted polymersomes. In addition, ds-DNA was found to be localized both in the nucleus and in the cytosol. The incubation of luciferase transfected B16-F10 cells with targeted polymersomes loaded with luciferase and Hsp90 expression silencing siRNAs yielded 31% and 23% knockdown in target protein expression, respectively. KEYWORDS. pH responsive polymersomes, polymer vesicles, siRNA delivery, tumor targeting, site-specific delivery, folate mediated targeting.


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INTRODUCTION In recent years, advances in polymer science have enabled a wide variety of novel materials for pharmaceutical and biomedical applications to be synthesized. A few of these polymers have been used to produce promising colloidal drug delivery systems, capable of enhancing the therapeutic performance of molecules that possess poor biopharmaceutical properties, inconvenient pharmacokinetic profiles or low therapeutic index.1-5 The requisites of polymeric nanocarriers for drug delivery include systemic and local biocompatibility, physicochemical stability, prolonged residence in the bloodstream as well as “stealth properties”, to escape the host immune response and phagocytosis.6 The combination of functional and structural components assembled into ‘virus-scale’ objects can bestow drug carriers with suitable physicochemical, biopharmaceutical and pharmacokinetic requisites for enhanced therapeutic profiles.1 At a molecular level, many important biopharmaceutical properties of drug nanovehicles derive from their interactions with biological structures such as proteins, cellular membranes and extracellular matrix biopolymers.7 Accordingly, surface-displayed functionalities, which bind specific target receptors for selective homing and subsequent controlled drug release, can yield delivery systems that interfacially “cross-talk” with the biological environment. Such carriers can be potentially exploited for delivery of a variety of valuable biotech drugs, namely proteins and nucleic acids. However, although these classes of therapeutics offer enormous promise for the treatment of many diseases, chemical and structural fragility and poor pharmacokinetic properties are the main hurdles to their pharmaceutical exploitation.8, 9


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Typically, the clinical application of siRNA as anti-cancer therapy is limited by rapid in vivo degradation by ribonucleases (RNase). Furthermore, since siRNAs display their biological activity by association with the RNA-inducing silencing complex (RISC) located in the cytosol, the intracellular siRNA delivery represents an additional obstacle to the therapeutic exploitation of these molecules.10-13 So far, a number of colloidal carriers have been developed to protect siRNA from degradation and promote internalization into target cells.14, 15 Polycation copolymers, in particular, have been tailored to efficiently complex with siRNA, which resulted in improved oligonucleotide stability and cellular up-take. For example, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (hy-PEI-g-PCL-b-PEG) copolymers have been found to form siRNA/copolymer complexes with longer circulation time in the bloodstream than complexes obtained with unmodified 25 kDa PEI. As a consequence, the former resulted in longer elimination half-life and three-/four-fold AUC increase of siRNA than the latter.16, 17 Polymeric carriers can also protect siRNA in transit from the bloodstream to the intracellular compartment and guarantee quick escape from the endosomal/lysosomal “pitfall”, thus avoiding siRNA degradation in early endosomes, late endosomes and lysosomes, and achieving siRNA intracellular targeting.18 Stimuli sensitive “multi-modular polymers” containing chargeable groups that reversibly switch from hydrophobic to hydrophilic materials according to the pH changes in the physiological compartments have been exploited to prepare “smart” multi-stage vesicles, namely polymersomes, which protect siRNA from degradation and provide for timely drug release.19-23 In polymersomes, the copolymer chains are spatially disposed to form membranes constituted by packed hydrophobic blocks while the hydrophilic blocks form hydrophilic layers on the vesicle surface and in the inner aqueous lumen.24 The outer corona can prevent vesicle opsonization,


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complement activation and in turn the host immune response and clearance from the body.25-27 Stealth polymersomes can also be up-graded by surface decoration with targeting agents that selectively recognize cells and promote the vesicle up-take by active internalization processes. We have recently developed polymersomes from self-assembling block copolymers, which contain pH sensitive imidazolic side chains partially end functionalized with folic acid as a targeting moiety. Folate receptors are in fact overexpressed by several tumor cell lines, thus endowing folic acid derivatised drug delivery systems with tumor targeting properties.28 Preliminary studies showed that the pH-responsive and folate-targeted polymersomes were physically stable in the presence of serum proteins, a prerequisite for in vivo exploitation. They also were found to efficiently encapsulate double stranded DNA, which was released under physio-pathological conditions (pH 5-8). In order to verify the biopharmaceutical and biological performance of the multi-stage responsive polymersomes generated from the triblock copolymers, we demonstrate here the pHcontrolled association of the copolymers with DNA and the encapsulation of functional siRNA. We also evaluated the role of the pH response due to the imidazole-containing side-chains in aiding endosomal escape of the polymersomes. The silencing activity of siRNA sequences delivered by the block copolymers was shown by the inhibition of luciferase and Hsp90 expression on a transfected cancer cell model with upregulated folate receptors.

EXPERIMENTAL SECTION Materials N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethyl-ammonium salt (Fluorescein-DHPE) was purchased from VWR International PBI


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(Milan, Italy). Double strand DNA (ds-DNA) and cyanin-3 labelled ds-DNA (C3-ds-DNA) (19 nucleotides per strand) were obtained from Biomers.net GmbH (Ulm, Germany). The DNAladder for electrophoretic mobility shift assay was purchased from GeneOn GmbH (Germany). Luciferase GL3 double strand siRNA (ds-siRNA), 21 nucleotides per strand, D-001400-01-20), control ds-siRNA and transfecting agent DharmaFECT® 1 were provided by Fisher Scientific (Madrid, Spain). Heat shock protein (Hsp) 90α/β double strand siRNA (ds-siRNA) with a pool of 4 target-specific 19-25 base pair nucleotides (sc-35610) and control double strand siRNA-A were provided by Santa Cruz Biotechnology, Inc (Dallas, TX - USA). Hsp90 alpha antibodies were obtained from Abcam (Cambridge, UK). Primary mouse anti-α-tubulin monoclonal antibodies and secondary rabbit anti-mouse IgG (whole molecule)-peroxidase antibodies were obtained from Sigma Aldrich (Madrid, Spain). PVDF transfer 0.45 μm membranes for Western blotting were obtained from Thermo Scientific (Madrid, Spain). The ds-DNA intercalating agent GelRed® was purchased from SICHIM (Rome, Italy). Quant-iT™ RiboGreen® RNA Assay Kit for siRNA quantification and goat anti-mouse Alexafluor 488 labeled monoclonal secondary antibodies were acquired from Life Technologies S.A. (Madrid, Spain). Anti-folate receptor alpha monoclonal primary antibody from mouse (1 mg/mL) in PBS was provided by Enzo Life Sciences Inc. (Farmingdale, NY-USA). Protease inhibitor cocktail tablets were provided by Roche (Basel, Switzerland). All products for cell biology including Dulbecco's modified Eagle medium (DMEM), L-glutamine, trypsin, antibiotic and antimicotic solution, bicinchoninic acid, copper (II) sulfate solution, fetal bovine serum (FBS), phosphate saline buffer with and without Ca2+/Mg2+ and Greiner plastics for cell culture were obtained from Sigma-Aldrich (St. Louis, MO-USA). Chamber slides BD FalconTM for confocal microscopy were purchased from SACCO (Cadorago, Italy). Vectashield® mounting medium with 4,6-diamidine-2-phenylindole (DAPI)


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was provided by Vector Laboratories Inc. (Burlingame, CA-USA). Cell lines from human breast cancer (MCF-7) and from human cervical carcinoma (KB) were provided by the cell bank ATCC (Manassas, VA-USA). Luciferase transfected B16-F10-luc-G5 Bioware® Cell Line from mouse melanoma were obtained from Xenogen Corporation (Alameda, CA-USA). All aqueous solutions were prepared using deionized water (milliQ-grade, 0.06 µSiemens cm-1) obtained using a Millipore MilliQ system (Bedford, MA- USA). Salts for buffer preparation and paraformaldehyde were provided by Riedel-de-Haën (Seelze, Germany), Fluka Analytical (Buchs SG, Switzerland) and Sigma-Aldrich (St. Louis, MO-USA).

Copolymer synthesis Triblock copolymers mPEG1.9 kDa-b-poly(ImHeMA)67-b-poly(GMA)36 (mPEG43-pImHeMA67pGMA36), tBoc–NH–PEG3.5 kDa-b-poly(ImHeMA)20-b-poly(GMA)58 (tBoc-PEG80-pImHeMA20pGMA58) and folate–NH–PEG3.5kDa-b-poly(ImHeMA)20-b-poly(GMA)58 (Folate-PEG80pImHeMA20-pGMA58) reported in Scheme 1 were synthesized by RAFT polymerization according to the protocols reported in our previous work.28

Scheme 1. Chemical structure of triblock copolymers: mPEG43-pImHeMA67-pGMA36 (1), tBocPEG80-pImHeMA20-pGMA58 (2) and Folate-PEG80-pImHeMA20-pGMA58 (3).


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Electrophoretic mobility shift assay Complexes of ds-DNA/mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58 at fixed 90:10 mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58 w/w % ratio and 0.07:1, 0.36:1, 0.72:1, 1.44:1, 2.16:1, 3.6:1, 14.4:1 nitrogen/phosphorus (N/P) molar ratio were prepared by mixing 3 µL of 2.9 × 10-10 M ds-DNA solution in 80 mM citrate buffer, pH 5.0, with 7 µL of 0.043-8.6 mg/mL mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20pGMA58 solutions in 80 mM citrate, pH 5.0. The N/P ratio was calculated assuming the mPEG43pIm67-pGMA36 nitrogen content was 2.8 × 10-6 mols/mg and tBoc-PEG80-pImHeMA20-pGMA58 nitrogen content was 1.11 × 10-6 mols/mg. Nitrogen content to calculate the N/P ratio in copolymers was referenced to the single protonatable nitrogen in the imidazole moiety. The ds-DNA/copolymer mixtures were electrophoretically run in a 12 % polyacrylamide gel. One well was loaded with 6 µL of 0.5 mg/mL DNA-Ladder in 10 mM Tris-HCl, 1 mM EDTA, pH 7.6. ds-DNA and 90:10 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20pGMA58 mixture were run as references. An aliquot (5 µL) of loading buffer containing 0.25% w/v bromophenol blue, 0.25 w/v % xylene cyanol and 30 w/v % glycerol in water were added to the samples in the wells and then the gel was run with 80 mM citrate, pH 5.0, at 100 mV for 1 hour and finally dipped for 1 hour in a staining medium obtained by diluting Gel Red® with mQ water. The gel was imaged using a UV-Transilluminator. Similarly, gel electrophoresis was performed using 45 mM Tris-borate, 1 mM EDTA, pH 7.4 as running buffer. In such a case the 90:10 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80pImHeMA20-pGMA58 sample was prepared by dissolving the copolymers in PBS (20 mM phosphate buffer, 154 mM NaCl), pH 5.0, and the pH was increased to 7.4 with 0.1 N NaOH.


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Polymersome preparation Copolymer solutions (1 mg/mL) were prepared by dissolving mPEG43-pImHeMA67-pGMA36, 90:10 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58 mixture and 90:5:5 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58/Folate-PEG80pImHeMA20-pGMA58 mixture in PBS, pH 5.0. The pH was then raised to pH 7.4 with 0.1 N NaOH to induce the copolymer self-assembling and polymersome formation.

ds-DNA and ds-siRNA polymersome loading All buffers used to prepare ds-DNA and ds-siRNA loaded polymersomes were autoclaved and filtered using 0.22 µm cut-off filters before use. ds-DNA loading. Aliquots (296 µL) of 300 µM cyanin-3 labelled ds-DNA (C3-ds-DNA) in 10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.8, were added to 500 µL of 2 mg/mL 90:10 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58 mixture or 90:5:5 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58/Folate-PEG80pImHeMA20-pGMA58 mixture in PBS, pH 5 to yield 0.78:1 N/P ratio. The samples were diluted to 1 mL with the same buffer and the pH was raised to 7.4 with 0.1 N NaOH. Non-encapsulated C3-ds-DNA was removed by dialysis using a 100 kDa MW cut-off Float-A-lyzer® system. The dialysis was performed for 24 hours using PBS, pH 7.4 as releasing buffer. An aliquot of the residual volume was acidified to pH 5.0 with 0.1 N HCl and C3-ds-DNA was determined by fluorescence spectroscopy (λex 550, λem 570).28 ds-siRNA loading. Polymersomes loaded with luciferase GL3 ds-siRNA and Hsp90α/β dssiRNA and with a scrambled sequence of ds-siRNA (negative control) were prepared by adding 80 µL of 100 µM ds-siRNA in 60 mM KCl, 0.2 mM MgCl2, 6 mM HEPES, pH 7.5, to 500 µL of


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2 mg/mL 90:10 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58 or 90:5:5 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58/ Folate-PEG80pImHeMA20-pGMA58 mixtures in PBS, pH 5.0. The volumes were raised to 1 mL with the same buffer to yield 1 mg/mL copolymer concentration and an N/P average molar ratio of 7.6:1. The pH was raised to 7.4 with 0.1 N NaOH to induce copolymer assembling and the nonencapsulated ds-siRNA was removed by dialysis as reported above. The amount of encapsulated ds-siRNA was determined using black 96 well plates. Volumes of 170 µL of 1 and 0.5 mg/mL mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58 and mPEG43-pImHeMA67pGMA36/tBoc-PEG80-pImHeMA20-pGMA58/Folate-PEG80-pImHeMA20-pGMA58 polymersomes loaded with ds-siRNA in PBS, pH 7.4, were spiked into the plate wells and added of 10 µL of 0.5 N HCl and 10 µL of Quant-iT™ RiboGreen® intercalating agent that was 200-fold diluted in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. Polymersomes only (i.e. no added ds-siRNA) at the same concentration of copolymers in the test samples were used as reference samples. The plates were incubated at room temperature for 3 minutes and then 10 µL of 0.5 N NaOH were added and the mixture analyzed by fluorescence (λex 485 nm/ λem 530 nm) using a microplate reader Wallac 1420 Victor2 Multilabel Counter from Perkin Elmer (Northwolk, CT, EEUU). The dssiRNA concentration was derived by subtracting the fluorescence emission of the copolymer alone (reference) from the emission intensity obtained with the samples of ds-siRNA loaded polymersomes. The ds-siRNA concentration in the samples was derived from a calibration curve obtained with known ds-siRNA concentrations in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5.

DLS, zeta potential and TEM analysis. Samples (1 mL) of 1 mg/mL polymersomes were prepared in 20 mM phosphate, 0.15 M NaCl at pH 7.4. The pH was adjusted with 0.5 N HCl.


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The mean size and polydispersity were measured by dynamic light scattering (DLS) analyses performed at 25 and 37°C using a Malvern Zetasizer NanoZS (Malvern Instruments Ltd., UK) supported by Zetasizer Software (version 6.12). Polymersomes (1 mg/mL) were analyzed by transmission electron microscopy (TEM). Samples were observed in negative staining mode, using small copper grid (400 mesh), covered by a "holey film" carbon layer. Samples were deposited on the grids and the contrast staining was performed with a uranyl acetate solution 1% w/v.

Copolymer hemolytic activity In tubes, 5 mL of heparinized mouse blood were diluted with 107.5 mL PBS, pH 7.4. The diluted blood was gently top-down shaken for thirty seconds and centrifuged at 3000 rpm for 10 minutes at 4°C. The buffer was removed and the blood cells were resuspended in the same buffer. The process was repeated twice and the diluted blood was divided into three volumes. After centrifugation, the pellet was suspended in PBS, pH 7.4 and 5.5. In 96 well plates, 100 µL of 0.5, 1, 2, 3 mg/mL of 90:10 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20pGMA58 polymersomes in PBS at pH 7.4 or 5.5, were added to 100 µL of the blood cell samples. The plates were incubated at 37°C for 1 hour and then centrifuged at 3000 rpm for 10 minutes at room temperature. The supernatants (100 μL) were transferred to a second plate and the absorbance of the released hemoglobin was measured at 570 nm with a microplate reader. Dextran (80 kDa) was used as negative control, whereas polyethylenimine (PEI) was used as a positive polymer control for hemolysis.29-32 1% w/v Triton X-100 was employed as a positive control reference (100% hemolysis).


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Cell culture KB cells (human cervical carcinoma) were grown at 37 °C, in 5 % CO2 atmosphere, using folic acid-free DMEM medium supplemented with 15 % FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL of amphotericin B. MCF7 cells (human breast adenocarcinoma) were grown at 37 °C, in 5 % CO2 atmosphere, using RPMI-1640 medium supplemented with 10 % FBS, 100 IU/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL of amphotericin B. B16-F10-luc-G5 cells from mouse melanoma and overexpressing folate receptor33, 34 were grown at 37 °C, in 5 % CO2 atmosphere, using DMEM medium supplemented with 10 % FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin.

Folate receptor expression study in KB and MCF7 cells KB and MCF7 cells were seeded in 25 cm2 cell culture flasks at a density of 5 × 104 cells/cm2 and grown for two days at 37 °C and 5 % CO2. KB cells were grown both in DMEM containing folic acid and in folic acid free DMEM as above reported. Medium was then removed, cells washed with PBS and detached from the flasks by scraping. Cells were pipetted and transferred in tubes for flow cytometric analysis and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed and the resulting cell pellet was collected (total volume of 100 µL). Primary antibody (2 µl of reagent solution) for folate receptor detection in PBS was added to the pellets, samples were mildly shaken for thirty seconds and incubated at room temperature for 5 minutes. Then, 2 µL of goat anti-mouse Alexa fluor 488 labeled monoclonal secondary antibody in PBS were added to cell samples and incubated at room temperature in the dark for 5 minutes. After incubation, samples were centrifuged at 1000 rpm for 5 minutes and repeatedly washed with


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PBS to remove the unbound secondary antibody and resuspended in 300 µL of PBS. Untreated cells were also prepared while the controls were generated by treating the cells with the secondary antibody only. Samples were analyzed by flow cytometry using λex and λem of 499/519 nm respectively using a FACS Diva flow cytometer (Becton Dickinson Inc., USA).

Effect of copolymers on cell metabolic activity. The effects of mPEG43-pImHeMA67-pGMA36, tBoc-PEG80-pImHeMA20-pGMA58 and FolatePEG80-pImHeMA20-pGMA58 on the metabolic activity of cells were evaluated by the MTS (3(4,5-dimethylthiazol-2-yl)- 5- (3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay using B16-F10-luc-G5 KB and MCF-7 cells. Cells were seeded in 96-wells plate at density of 10,000 cells/well and treated with 200 µL of vesicle suspensions in PBS at pH 7.4 at increasing concentrations [1, 2 and 3 mg/mL]. Polymersomes were obtained by the “pHswitch” technique with the following copolymers ratios: a) 90:10 w/w % mPEG43-pImHeMA67pGMA36 / tBoc-PEG80-pImHeMA20-pGMA58 mixtures; b) 90:5:5 w/w % mPEG43-pImHeMA67pGMA36 / tBoc-PEG80-pImHeMA20-pGMA58 / Folate-PEG80-pImHeMA20-pGMA58 mixture. After 24 and 48 h incubation, 10 µL of a 20:1 (v/v) mixture of 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium/phenazine methosulfate (MTS/PMS; 2 mg/mL MTS, 0.92 mg/mL PMS) were added to each well and the plate was incubated for 2 hours at 37 °C. The absorbance was measured at 496 nm using a microplate reader. The extent of metabolic activity change as a proxy for cell viability was expressed as the percentage referred to untreated cells.


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Cell up-take studies. Flow cytometry study. A kinetic up-take study of folate targeted vesicles loaded with labeled dsDNA was performed using B16-F10-luc-G5 cells by evaluating the cell association of the vesicles over time. Cells were seeded in a 6 well plate at a density of 31 × 103 cells/cm2. The medium was then removed, cells were washed twice with PBS and 1 mL of a 1 mg/mL dispersion of C3-ds-DNA loaded polymersomes in PBS, pH 7.4 obtained with a 90:5:5 w/w % of mPEG43-pImHeMA67-pGMA36 / tBoc-PEG80-pImHeMA20-pGMA58 / Folate-PEG80pImHeMA20-pGMA58 and with a 90:10 w/w% of mPEG43-pImHeMA67-pGMA36 / tBoc-PEG80pImHeMA20-pGMA58 was added to the wells. Cells were incubated at 37 and 4 °C. After 0, 15, 30 minute, 2 hour and 5 hour incubation time, wells were washed with PBS and cells were detached by scraping the suspensions on ice and transferring the contents into cytometer tubes. The samples were redispersed in 300 µL of PBS and analyzed by flow cytometry using a BD FACSDiva flow cytometer Cytomics FC 500 (Beckman Coulter Inc.) (λex 550, λem 570). Data collection involved 15,000 counts per sample and was analyzed by using the percentage of positive cells. A study was performed using KB and MCF7 cells to quantify the cell up-take of C3-ds-DNA loaded polymeric vesicles. An incubation time of 30 minutes at 37 °C was selected for this study. After incubation, the copolymer dispersions were discharged and wells washed with PBS. To detach cells from the plastics, cells were washed with PBS and treated as before by scraping on ice prior to transfer to cytometer tubes. Samples were redispersed in 300 µL of 1 % w/v freshly prepared paraformaldehyde (PFA) in PBS and analyzed by flow cytometry as reported above. Confocal microscopy. Glass BD Falcon™ chamber slides with 1.7 cm2 growth surface area per well, were pretreated with a sterile poly-D-lysine hydrobromide solution (0.2 mg/mL) dissolved


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in sterile mQ water to increase cell attachment. Chamber slides were incubated with the poly-Dlysine solution for 1 hour at room temperature in the safety cabinet and the solution was then removed. Wells were rinsed three times with PBS and KB and MCF7 cells were seeded at a density of 59 × 103 cells/cm2 and grown for 24 h at 37 °C and 5 % CO2. The medium was then removed, cells were washed with PBS. 500 µL of C3-ds-DNA loaded polymersomes at concentration of 1 mg/mL were prepared with 90:5:5 w/w % of mPEG43-pImHeMA67-pGMA36 / tBoc-PEG80-pImHeMA20-pGMA58/Folate-PEG80-pImHeMA20-pGMA58 (targeted polymersomes) and 90:10 w/w% of mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20pGMA58 copolymer mixture (non-targeted polymersomes) in PBS at pH 7.4 as previously described and were added to each well. Cells were incubated at 37 °C in the dark for 30 minutes. Polymersome suspensions were then removed and wells were gently washed three times with PBS. Cells were fixed with 500 µL of freshly prepared 1 w/v % PFA in PBS for 20 minutes at 4 °C, washed with PBS and incubated with 200 µL of a 5 µg/mL fluorescein-DHPE solution in PBS for 10 minutes in the dark to visualize the cell membranes. Cells were washed three times with PBS, chamber slides were disassembled and 20 µL of Vectashield® mounting medium containing DAPI for nuclei staining were added to each slide. Finally, slides were covered with coverslips. Samples were analyzed by confocal microscopy using an immersion lens with 63X magnification. Lasers with emission wavelengths at 405, 488 e 561 nm were used to detect DAPI, fluorescein-DHPE and cyanine-3 labeled ds-DNA.

siRNA mediated silencing study. B16-F10-luc-G5 cells33, 34 were seeded in a 96 well plate at a density of 31 × 103 cells/cm2. 100 µL of a 1 mg/mL suspension of luciferase silencing ds-siRNA loaded copolymer assemblies


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obtained with 90:5:5 w/w % of mPEG43-pImHeMA67-pGMA36 / tBoc-PEG80-pImHeMA20pGMA58 / Folate-PEG80-pImHeMA20-pGMA58 in PBS at pH 7.4 were added to the wells and incubated at 37 °C for 30 minutes. Cells were also treated with luciferase silencing ds-siRNA complexed with the transfecting agent DharmaFECT® 1, scrambled ds-siRNA complexed with DharmaFECT® 1, targeted polymersomes loaded with a scrambled ds-siRNA sequence, nonformulated luciferase silencing ds-siRNA and non-formulated scrambled ds-siRNA as controls. DharmaFECT® 1 agent was used at a final 3.3 µg/mL concentration according to manufacturer instructions. After incubation, copolymer dispersions were discharged and replaced with DMEM medium supplemented with 10 % FBS. Cells were grown for 24 and 48 h. Afterwards, 16 µL of luciferin (150 µg/mL) in PBS was added to each well and the Luciferase activity was spectrofotometrically quantified using a microplate reader at λem 535 nm. B16-F10-luc-G5 cells were also seeded in 6 well plates to study the expression of Hsp90. Aliquots (100 µL) of 1 mg/mL of Hsp90 silencing ds-siRNA loaded copolymer assemblies obtained with 90:5:5 w/w % of mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20pGMA58 / Folate-PEG80-pImHeMA20-pGMA58 in PBS at pH 7.4 were added to the wells, and incubated at 37 °C for 30 minutes. Cells were also treated with targeted polymersomes loaded with a scrambled ds-siRNA sequence, Hsp90 ds-siRNA and scrambled ds-siRNA complexed with transfecting agent DharmaFECT® 1 as controls. DharmaFECT® 1 agent was used at a final 3.3 µg/mL concentration according to manufacturer instructions. After incubation, copolymer dispersions were discharged and replaced with DMEM medium supplemented with 10 % FBS. Cells were grown for 24 hours. Afterwards, plates were transferred on ice, the medium was discharged and cells washed twice with cold PBS. Then, 100 µL of lysis buffer (50 mM TrisHCl pH 8, 120 mM NaCl, 0.02 w/v % NaN3, 0.1 w/v % SDS, 1 v/v% Nonidet P-40, 0.5 w/v %


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deoxycholic acid, 1x protease inhibitor cocktail) were added and cells were scraped off to obtain a cell suspension that was transferred into microtubes. Afterwards, the tubes were centrifuged at 4 °C, 10,000 rpm for 10 minutes, and the protein concentrations in the supernatant fraction were determined using Bio-Rad Protein Assay (Bio-Rad Laboratories, CA, USA). For western blotting, samples containing 25 µg of total proteins were loaded onto 8 % polyacrylamide gels. After electrophoresis, the proteins were then transferred to nitrocellulose membranes. Then, membranes were blocked in 5 % of non-fat dry milk (NFDM) diluted in 0.05 v/v % Tween 20 in water for 1 hour and incubated overnight at 4 °C with mouse monoclonal Hsp90 alpha primary antibody and mouse monoclonal anti-α-tubulin primary antibody (2 mg/mL). Excess antibody was then removed by washing the membrane in PBS containing 0.1 v/v % Tween 20, and the membranes were incubated for 2 h with rabbit Anti-Mouse IgG (whole molecule)-Peroxidase secondary antibody (10-20 mg/mL). After incubation, the membranes were washed with 0.1 v/v % Tween-20 in PBS for three times and immune detection was performed with the use of the chemiluminiscent (ECC) western blotting detection system (Amershan Bioscience) according to the manufacturer’s instructions.

RESULTS Scheme 1 reports the structure of the triblock copolymers used in this study: 1. mPEG43pImHeMA67-pGMA36 (23,900 g/mol), 2. tBoc-PEG80-pImHeMA20-pGMA58 (17,900 g/mol) and 3. Folate-PEG80-pImHeMA20-pGMA58 (18,300 g/mol).


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Oligonucleotide/copolymer association Electrophoretic chromatography was performed to evaluate the association of oligonucleotides with the copolymers. The study was performed using ds-DNA as oligonucleotide model and 90:10 w/w % of mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58 mixture. Copolymer mixtures were incubated with ds-DNA in order to yield N/P molar ratios ranging from 0.07:1 to 14.4:1. Figure 1 reports the gel electrophoretic migration profiles obtained with the various copolymers/ds-DNA molar ratios and the migration of the references: ladder, dsDNA alone (copolymer free ds-DNA) and copolymers alone (ds-DNA free copolymer mixtures). At pH 5.0 (Figure 1A), ds-DNA was clearly detected in the ds-DNA reference and in samples containing copolymers/ds-DNA at 0.07:1-1.44:1 N/P molar ratio. The samples containing copolymers/ds-DNA at 2.16:11 N/P molar ratio showed a light band corresponding to free dsDNA while no ds-DNA was detected in the samples containing copolymers/ds-DNA at 3.6:1 and 14.4:1 N/P ratio samples. The absence of detectable ds-DNA in samples containing copolymers/ds-DNA at 3.6:1 and 14.4:1 N/P molar ratio indicates that in these samples ds-DNA is completely associated with the copolymers, which shield ds-DNA from staining by the GelRed™ Nucleic Acid Gel Stain as reported in the literature.35-37


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A

B

Figure 1. Electrophoretic mobility profiles of ds-DNA in the presence of 90:10 w/w % mPEG43pImHeMA67-pGMA36/ tBoc-PEG80-pImHeMA20-pGMA58 mixtures at pH 5.0 (A) and pH 7.4 (B).


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Figure 1B shows that at pH 7.4 the migration of ds-DNA is not significantly affected by the N/P feed ratio, and very little copolymer/oligonucleotide complexation occurred under this analytical condition.28

Red blood cell interaction pH responsive polymersomes prepared by “pH-switch” technique were incubated with red blood cells (RBC) to test their ability to disrupt cell membranes, as a proxy measure of potential to undergo endosomal escape.38 Dextran was tested as negative control. Polyethylenimine (PEI) was chosen as positive control for its known hemolytic activity.31, 32 Figure 2 shows the hemolytic profiles obtained by RBC incubation with 90:10 w/w% mPEG43pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20-pGMA58 polymersomes at the different pHs.


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Hemolysis (%)

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Figure 2. Hemolytic profiles of 90:10 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80pImHeMA20-pGMA58 polymersomes (♦), PEI (▲), dextran (■) at: A. pH 7.4; B. pH 5.5. Data are represented as mean values ± SD, with n= 3 experiments.

Figure 2A shows that at pH 7.4, which mimics the conditions present in the bloodstream, the imidazole-based pH responsive polymersomes do not display hemolytic activity at all tested concentrations. In contrast, PEI was found to be membrane-active at this pH. Figure 2B shows that at pH 5.5, which mimics the endosomal compartment during digestive process, the copolymers induced remarkable RBC membrane rupture even at low concentrations. About 70% of hemolytic activity was indeed observed with 0.5 mg/mL copolymer concentration, which was much higher than the rupture induced by PEI at the same pH and concentration.


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Polymersomes siRNA loading and characterization Similarly to plain polymersomes, ds-siRNA loaded polymersomes were prepared according to the “pH-switch” technique. Non-targeted (non-folated) and targeted (folated) polymersomes were prepared by using 90:10 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20pGMA58 and 95:5:5 w/w % mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20pGMA58/Folate-PEG80-pImHeMA20-pGMA58, respectively, and 7.6:1 N/P molar ratio. The N/P molar ratio was selected according to the electrophoretic data, which showed that complete oligonucleotide association to the copolymers at pH 5.0 required an N/P feed ratio above 3.6:1. The size distribution profile of ds-siRNA loaded polymersomes reported in Figure 3 was determined by Dynamic Light Scattering (DLS) analysis at 25 °C after dialysis. ds-siRNA loaded non-targeted and targeted polymersomes possessed mean diameters of 273 ± 11 nm (PDI = 0.102) and 173 ± 7 nm (PDI = 0.082), respectively. Spherical objects were observed by TEM imaging of the polymer ds-siRNA formulations at pH 7.4.


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B

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00

500 nm

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500 nm

Figure 3. DLS size profiles (A and C) and TEM images (B and D) of non-targeted ds-siRNA loaded polymersomes (A and B) and targeted ds-siRNA loaded polymersomes (C and D).

The ds-siRNA loading in the polymersomes determined by fluorescence analysis using QuantiT™ RiboGreen® intercalating agent showed a loading capacity (LC) of 5.4 mol/mol % dssiRNA/copolymer, which corresponds to 3.1 w/w % ds-siRNA/copolymer.

Folate receptor expression in KB and MCF-7 cell lines Cell phenotypic studies were performed to investigate the expression of folate receptors on the cell membrane of human cervical carcinoma KB cells grown in folic acid free or folic acid supplemented media. The breast cancer MCF-7 cells were grown in folic acid supplemented media. The cell phenotypic profiles were evaluated by tagging live KB and MCF-7 cells with specific anti-folate receptor antibody conventionally used in immunohistochemical settings, and


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a secondary fluorescent antibody as labeling agent. The labelled cells were then analyzed by flow cytometry. The flow cytometry data of KB and MCF-7 cells reported in Figure 4 show that KB cells highly expressed the folate receptor since almost 100 % of cells tested positive in the receptor labelling procedure. Furthermore, the growth medium did not significantly affect the expression of the folate receptor in KB cells. Indeed, Figures 4A and 4B show that the receptor expression was not increased when cells were grown in folic acid free medium with respect to cells grown in folic acid containing DMEM medium.

A

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Figure 4. KB and MCF-7 cell cytometric profile obtained after cell treatment with the secondary antibody only (■, control untreated sample) and after treatment with anti-folate receptor antibody and secondary antibody (■). A. KB cells grown in folic acid supplemented DMEM medium; B. KB cells grown in folic acid free DMEM medium; C. MCF-7 grown in folic acid supplemented RPMI medium.


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Figure 4C shows that in the case of the MCF-7 cell line, only 13% of the cell population was positive for the folate receptor labelling indicating that this cell line could be used as a low folate receptor expressing cell control in the cell interaction and up-take studies.

Effects of polymersomes on cell metabolic activity The cytocompatibility of the non-targeted and targeted polymersomes was examined by incubating increasing concentrations of polymersomes with folate receptor overexpressing KB cells, and low folate receptor expressing MCF-7 cells. Folate receptor overexpressing B16-F10 mouse melanoma cells33, 34 were also used in this study. The metabolic activity of the cells was determined by a standard MTS assay.39

The cell viability data reported in Figure 5 show that both the non-targeted and targeted polymersomes were well-tolerated by the cell lines even at high concentrations.


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Figure 5. MTS cell metabolic activity profile of B16F10 (■), KB (■) and MCF-7 (■) cells incubated with of 90:10 w/w % of mPEG43-pImHeMA67-pGMA36/tBoc-PEG80-pImHeMA20pGMA58 polymersomes (A and B) and 90:5:5 w/w % of mPEG43-pImHeMA67-pGMA36/ tBocPEG80-pImHeMA20-pGMA58/Folate-PEG80-pImHeMA20-pGMA58 polymersomes (C and D). The cells were incubated with the polymersomes for 24 (A and C) and for 48 hours (B and D). Data are reported as mean values ± SD, with n=5 experiments.

Flow cytometric analysis Kinetic up-take study. Kinetic up-take studies were initially undertaken to set the experimental conditions for the biological investigations.


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Cyanine-3 labelled ds-DNA (C3-ds-DNA) loaded folate-tipped polymersomes were incubated with B16-F10 cells at 37 and 4 °C and then analyzed by flow cytometry. The study at 4 °C was carried out to assess whether the cell internalization mechanism was energy-dependent.40 Figure 6 shows the time dependent up-take profile represented as percentage of fluorescently positive cells. In order to calculate the fraction of up-take associated with energy-dependent process, i.e. specific receptor-mediated pathways, the fluorescence values obtained with polymersomes at 37 °C were subtracted from the fluorescence ascribable solely to the adsorption of the polymersomes on the cell membrane obtained by incubating cells with the polymersomes at 4 °C. The cell up-take profiles described in Figure 6A show that the cells treated with non-targeted polymersomes display negligible fluorescence, either at 4 or 37 °C, indicating that these polymersomes do not undergo significant cell association.


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Figure 6. B16-F10 cell kinetic association profile of control non-targeted polymersomes (A) and targeted polymersomes (B) loaded with C3-ds-DNA at 37 °C (■) and 4°C (♦). Positive cell percentage values obtained by subtracting the data obtained at 37 °C of the data obtained at 4ºC are also reported (▲). Data represented as mean values ± SD, with n = 3 experiments.

The results reported in Figure 6B show that the maximal internalization of the targeted polymersomes at 37 °C occurred after 30 min incubation. Afterwards, the up-take decreased, in agreement with studies performed with folate modified macromolecules previously reported.41


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Comparative up-take study. Based on the outcome of the kinetic up-take study with B16-F10 cells, targeted and non-targeted polymersomes were incubated both with KB and MCF-7 cells for 30 minutes to investigate the capacity of the vehicle to deliver oligonucleotides to cancer cells by active targeted mechanisms. Figure 7 shows the fluorescence profiles obtained by KB and MCF7 cell incubation with non-targeted and targeted polymersomes. Figure 8 reports the percentage of positive cells for the cell samples calculated by quantification of the results obtained by flow cytometric analysis. A

B

Figure 7. Cell up-take flow cytometry study of C3-ds-DNA loaded targeted polymersomes and non-targeted polymersomes in MCF-7 (A) and KB (B) cells. C3-ds-DNA loaded targeted polymersomes (solid line) and non-targeted polymersomes (dashed line), and untreated cells (■).


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Positive cells (%)

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*

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Figure 8. Percentage of positive KB and MCF-7 cells incubated with polymersomes loaded with C3-ds-DNA obtained by the elaboration of the flow cytometry data. Cells were treated with targeted polymersomes (■) and non-targeted polymersomes (■). Data are represented as mean values ± SD, with n = 3 experiments.

The results described in Figure 8 show that the targeted and the non-targeted polymersomes were internalized by less than 20 % of the MCF7 cells, in agreement with the relatively low proportion of cells expressing the folate receptor. In the case of the folate receptor overexpressing KB cells, up-take of non-targeted polymersomes was observed on ~ 10 % of cells, whilst a significantly higher up-take (> 50% of cells) occurred for the targeted polymersomes. This indicated that the folate moieties on the polymersome surface promoted the internalization of polymersomes into cells that expressed higher levels of the folate receptor. The flow cytometric analysis showed that the KB cell up-take of folate targeted polymersomes was 5.5-fold higher with respect to the non-targeted polymersomes. On the contrary, no significant difference in the cell association was measured with MCF-7 cells, which showed overall low association. Furthermore, the up-take of the nanosystems was higher in KB cells


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compared to MCF-7 cells, and the highest overall up-take was observed for the copolymer formulations containing 5 w/w % of the folate-tipped Folate-PEG80-pImHeMA20-pGMA58.

Confocal microscopy. Confocal microscopy investigations were performed by incubating KB and MCF-7 cells for 30 minutes with targeted and non-targeted cyanine-3 labeled ds-DNA (C3-ds-DNA) loaded polymersomes (Figure 9).

A

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Figure 9. Confocal microscopy images of cells treated with polymersomes. Images were acquired with a 63X oil immersion objective. KB cells incubated with: A. non-targeted C3-dsDNA loaded polymersomes; B. folate targeted C3-ds-DNA loaded polymersomes. C is a


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magnification (252X) of the inset area of panel B showing details of KB cell nucleus and intracellular fluorescent compartments. MCF7 cells incubated with: D. non-targeted C3-ds-DNA polymersomes; E. folate targeted C3-ds-DNA polymersomes. Cell images were acquired using a blue channel for nuclei detection after labelling with DAPI, a green channel for cell detection after labelling with fluorescein-DHPE and a red channel for C3-ds-DNA detection.

The confocal microscopy images reported in Figure 9 confirmed the results obtained by flow cytometric analysis. In the case of KB cells treated with non-targeted polymersomes, and MCF-7 cells incubated either with targeted and non-targeted polymersomes, localization of C3-ds-DNA (red fluorescence) was neither observed in the cytosol nor in the nucleus. On the contrary, in the case of KB cells treated with targeted polymersomes, C3-ds-DNA was efficiently and selectively internalized. Furthermore, the images reported in Figure 1B and its magnification (Figure 1C) show discrete punctate red fluorescence in the cytosol and in the nucleus of the KB cells, attributable to C3-ds-DNA associated polymersomes. The magnified image also highlights that the fluorescence from C3-ds-DNA was either associated to confined cytosolic sub-compartments or spread throughout the cytosol.

In vitro silencing studies Luciferase inhibition. Luciferase silencing ds-siRNA, empty targeted polymersomes and targeted polymersomes loaded with luciferase silencing ds-siRNA or scrambled ds-siRNA were incubated with B16-F10-luc-G5 cells from mouse melanoma.42 The bioluminescence emission of cell samples was spectrophotometrically monitored at 535 nm.


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80

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Figure 10. Luciferase activity in B16-F10-luc-G5 cells treated with, from the left: scrambled dssiRNA formulated with DharmaFECT® 1, luciferase silencing ds-siRNA formulated with DharmaFECT® 1, targeted polymersomes, targeted polymersomes loaded with scrambled dssiRNA, targeted polymersomes loaded with luciferase silencing ds-siRNA, free scrambled dssiRNA, free luciferase silencing ds-siRNA. Data are represented as mean values ± SD, with n = 5 experiments. Significant differences (p < 0.05) compared to controls are depicted with an asterisk.

The bioluminescence results reported in Figure 10 show that after 30 minutes incubation, the targeted polymersomes loaded with luciferase silencing ds-siRNA reduced the luciferase activity by 31%. No silencing effect was observed when the cells were incubated with empty targeted polymersomes or with targeted polymersomes loaded with scrambled ds-siRNA. Furthermore, no biological activity was shown by free anti-luciferase ds-siRNA (not loaded in polymersomes).


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The positive control obtained by incubating cells with luciferase silencing ds-siRNA formulated with DharmaFECT® 1 showed a silencing outcome of 60%. Hsp90 inhibition. Targeted polymersomes loaded with a functional ds-siRNA for silencing Hsp90 gene expression were incubated with luciferase-expressing B16-F10-luc-G5 cells. Figure 11 shows the expression of Hsp90 proteins quantified by “ImageJ” analysis software. As a control, the expression of α-tubulin was monitored to ensure equal loading and transfer of proteins. The protein expression bands, from cells treated with Hsp90 silencing ds-siRNA formulated with commercial transfecting agent (Fig 11A, lane c) and with targeted polymersomes reported in Figure 11A, lane f, were less intense with respect to the protein bands obtained with the other formulations and controls. Figure 11B shows the Hsp90 inhibition profile obtained by the Western Blot analysis.

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Figure 11 A. Effect on Hsp90 expression after the incubation of Hsp90 silencing ds-siRNA formulations. B. Relative Hsp90 expression in cells after incubation with scrambled ds-siRNA and Hsp90 silencing ds-siRNA transfected with: B1. DharmaFECT® 1; B2. targeted polymersomes. Data are represented as mean values ± SD, with n = 3 experiments. Significant differences (p < 0.05) are shown with an asterisk.


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The cells treated with Hsp90 silencing ds-siRNA loaded targeted polymersomes reduced the protein expression by 23 %. Inhibition of 37 % in Hsp90 expression was achieved with Hsp90 silencing ds-siRNA with the commercially available transfecting agent DharmaFECT® 1 used as positive control. Similarly to what was observed for the luciferase silencing study, no evidence of silencing effect was detected when cells were incubated with empty targeted polymersomes and scrambled ds-siRNA loaded targeted polymersomes.

DISCUSSION The triblock PEG-pImHeMA-pGMA based copolymers described in the present work were designed to produce colloidal systems for oligonucleotide selective delivery to biological targets through conformational changes triggered by microenvironmental features of tumor interstitia and intracellular compartments.28 Previous studies showed that copolymers bearing two hydrophilic terminal blocks, polyethylenglycol (PEG) and polyglycerolmethacrylate (polyGMA), and an ionizable polyC6-imidazole-methacrylate internal block, exhibited pH dependent self-assembling properties. The size and shape of the resulting colloidal structures and importantly their ‘interfacing’ features (i.e. the functionality displayed at surfaces) were shown to respond dynamically and reversibly to micro-environmental stimuli. Copolymers containing imidazole moieties are commonly found in biology as they have been selected for their ability to undergo protonation/deprotonation over a narrow pH range. Typically, the pH-sensitivity of histidine governs the role of the catalytic triad of proteases. It is worth noting that the pKa values of imidazole rings in biopolymers, namely histidine residues in proteins, and in synthetic polymers is close to the pH ranges observed in pathophysiological conditions. In particular,


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hypoxic and glycolytic regions of tumor matrices as well as subcellular digestive compartments display pH ranges of ~ 5-7. In addition, the pKa values of imidazole groups can be fine-tuned by other functional groups conjugated to and/or surrounding the heterocyclic rings.43 Accordingly, polyimidazole derivatives are expected to change protonation state over these pH ranges. When coupled to a drug carrier, these chemical moieties can elicit pH triggered conformational changes in the carrier, which can result in altered interaction with biological structures, namely cell surfaces, or boost the drug release in target sites. For this study we selected PEG-pImHeMA-pGMA block copolymers from a library of related materials as they were found to self-assemble into polymersomes at physiological pH (pH 7.4) and undergo structural alteration and dissociation at acidic pH (pH 6.5–5.0). This behavior suggested applications for drug delivery in specific biological compartments such as endosomes and lysosomes or in hypoxic tumor tissue.44 Furthermore, the colloidal nature of these polymersomes was intended to bestow drug carriers with suitable properties for passive localization in tumor tissues by the enhanced permeability and retention (EPR) effect. The synthetic route also enabled the resulting copolymers to be tagged easily with folic acid groups, which would be surface-displayed upon self-assembly into polymersomes, and which might aid recognition and internalization by folate receptors overexpressed by several tumor cells.45-47 In our previous study we selected the composition of the polymersomes to include 5 % of the folated copolymer and 5 % of tBoc-terminating copolymer in the system in order to yield stable polymersomes under physiological conditions, while maintaining the ability to undergo rearrangement and vesicle disassembly under pathophysiological conditions. However, it was found that these colloidal carriers behaved rather differently in the presence of oligonucleotides, compared to assembly in the formation of empty polymersomes. Indeed, in the


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case of empty polymersomes, the polyimidazole block deprotonation at pH 7.4 yielded rapid vesicle formation whilst at lower pH the protonation of imidazoles caused disassembly and loosely associated free copolymers in solution. By contrast, the pH can both dictate the ionizable copolymer association into polymersomes and oligonucleotide association. The association study performed by electrophoretic analysis of PEG-pImHeMA-pGMA/ds-DNA mixtures showed that at pH 5.0 the polyimidazole of the PEG-pImHeMA-pGMA copolymers, which possessed an apparent pKa of 5.9 and was expected to be predominantly protonated under these conditions,48 formed imidiazole/phosphate (N/P) charge-to-charge complexes with oligonucleotides. The threshold for complete association was obtained at a 2.16:1 N/P molar ratio, which corresponded to ~2 imidazole groups per phosphate. No PEG-pImHeMA-pGMA/dsDNA association was observed at pH 7.4, which we attributed to the deprotonation of imidazole residues at higher pH. However, at this pH the imidazole group deprotonation would switch the polymidazole block from a hydrophilic to a hydrophobic state, thus driving the formation of polymersomes. Therefore, although the polyimidazole block deprotonation disfavored copolymer/oligonucleotide association, which takes place by polyimidazolium/oligonucleotide ion-pairing, the encapsulation of the non-complexed oligonucleotide within the water pool of the vesicle interior may have become favored. It has in fact proved possible to load these polymersomes with ds-DNA and ds-siRNA at physiological pH (7.4), even though at this pH the electrophoresis results suggested oligonucleotides and the polyimidazole block copolymer did not form complexes. These data can be rationalised by the hypothesis that preliminary formation of polymer/oligonucleotide complexes at pH 5.0 guide oligonucleotide encapsulation into the vesicle core, but in loose association with the copolymer assembled in vesicles that could be disrupted in the high field and shear conditions of electrophoresis.copolymer Therefore, the


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encapsulation of oligonucleotides within the polymersomes derives from a fine balance of interaction with charged imidazole blocks of the copolymers, which forms a vesicle membrane by a “hydrophobic multi-ion pair” interaction”. The increase in size of ds-siRNA loaded polymersomes with respect to the empty polymersomes was in line with that previously observed with ds-DNA loaded polymersomes and indicated that the entrapped or encapsulated polyanionic macromolecules modified the arrangement of the triblock copolymer chains within the overall structure of the colloidal carrier. The size of targeted and non-targeted siRNA loaded polymersomes is in agreement with data previously reported for dsDNA loaded polymersomes.28 The slight increase in size of targeted vesicles may be ascribed to rearrangements of chains containing the folated termini. Indeed the anionic and hydrophobic character of the terminal moiety might be expected to reduce the hydrodynamic radii of the polymersomes by charge-charge repulsion and hydrophobic compaction. The slightly lower vesicle loading obtained for ds-siRNA compared to the ones obtained with the ds-DNA28 was most likely due to the slightly different loading conditions with respect to those undertaken for the ds-DNA preliminary loading, namely lower copolymer/ds-siRNA feed ratio and concentration of the ds-siRNA during vesicle assembly. However, the overall results showed that the polymersomes were suitable for encapsulation of significant amounts (5.4 mol %) of oligonucleotides with molecular weight of about 13.3 kDa, namely ds-siRNA. The hemolytic study showed that PEG-pImHeMA-pGMA based polymersomes displayed a pH dependent interaction with biomembranes. The polymersomes were highly compatible with cell membranes under physiological conditions. By contrast, cell membrane disruption was observed


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to occur at acidic pH, suggesting that these polymersomes should have the capacity to escape endosomes, a requisite for delivery of siRNA in the active form to the cytosol. In prior studies, endosomal escape38 has been postulated to occur either due to the "proton sponge effect" of buffering polymers with an apparent pKa matching the pH of endosomes (5.5-6.5), or to the interaction of the charged polymers with endosomal membranes and their consequent destabilization.49 It is likely therefore that the entry of the polymersomes described here into endosomal compartments and a subsequent pH change might have resulted in strong membrane disruption, with the possibility of exit of the polymersomes with siRNA into the remainder of the cell interior. It is interesting to note that PEI displays a lower toxicity at pH 5.5 than at 7.4. This could be due to the partial deprotonation of secondary amino groups at pH 7.4 (pKa 6.7) that can increase the hydrophobicity of the polymer, which results in enhanced membrane interaction and damage. Biological studies to evaluate the selectivity of the polymersomes for entry into cancer cells were anticipated by an initial phenotypic characterization of specific cells for folate receptor expression. Studies of cell phenotypes have been carried out previously by tagging the folate receptor with radioactive labels or by Western blot assays on cell homogenates.50, 51 In the present study, folate receptor expression in two cancer cell lines, KB cells from human cervical carcinoma and MCF-7 cells from human breast adenocarcinoma, was characterized using flow cytometry. The advantage of this method compared to those reported in the literature lies in the detection of active membrane-associated folate receptor only, without the risk of detecting nonfunctional receptor proteins present in the cytosol. The results showed that KB cells under selected assay conditions expressed high levels of the folate receptor (> 99 % of cells were FRpositive), with a negligible effect of the culture medium on receptor expression level. The MCF-


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7 cell line used as a ‘negative’ control expressed the receptor to a much lower level (~13% of cells tested). The reduced level of the receptor in this cell line probably explains why MCF-7 cells do not grow well in folic acid free media.

Active targeting to cancer cells is a mandatory feature for carrier vehicles delivering biologically active oligonucleotides such as siRNA and miRNA to molecular targets within the cytosol (namely the multi-protein RNA-inducing silencing complex, RISC). Active targeting through the folate receptor promotes the endocytosis and the access to the cytosol of carriers and hydrophilic macromolecules, which are prerequisites for therapeutic efficacy, and simultaneously enhance site-selectivity towards cancer cells within the tumor tissue. In agreement with the hemolysis results obtained at pH 7.4, the initial cell up-take studies showed that the polymersomes were very well-tolerated by all tested cell lines as no significant cytotoxicity was detected after prolonged (48 h) cell exposure. Folate targeted polymersomes tested with KB cells showed significantly higher cell up-take compared to the control polymersomes, while the MCF-7 cells internalized to a lower extent the folate targeted polymersomes under the same conditions. This confirmed that the internalization process occurred primarily by an active mechanism through recognition of the cell surface receptors. Even though no unique consensus concerning the mechanism of cell uptake of folate decorated colloidal systems can be found in the literature, the cell up-take of the targeted polymersomes seems to occur through a receptor mediated mechanism according to a caveolae mediated52-55 or a clathrin-coated pit process56, 57. The kinetic up-take study, initially performed to set-up the experimental conditions for the cell internalization investigations, were in good agreement with the evidence that targeted polymersomes entered cells by an energy dependent


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process, which was mostly inhibited (~90 % after 30 min incubation) when cells were incubated at 4 °C.40 The kinetic profile of polymersomes entry by folate receptor overexpressing cells (B16-F10) was in very good agreement with the results reported by Leamon and Low for other folate-tagged carriers,58 with maximum internalization in 30 minutes. The slight internalization decrease observed after 30 minutes could be ascribed to folate receptor saturation. Flow cytometry and confocal microscopy data demonstrated that fluorescently labelled ds-DNA loaded polymersomes were present both in confined regions in the nucleus, and partially spread over the cytosol. The microscopic investigation confirmed the endosomolytic capacity of the polymeric vehicles, as the labelled ds-DNA had escaped the internalizing organelles and migrated to the cytosol first and then to the nucleus. The access of exogenous small oligonucleotide sequences delivered as polyplexes and lipoplexes to the nucleus has been recently demonstrated.59 Polyplexes were reported to have exited the endosomes while still in the polymer/oligonucleotide complex form, with de-complexation possibly occurring after a few hours when they had migrated in the cytosol and the nucleus.60 However, the migration of dsDNA in the copolymer associated state or as free macromolecule would clearly be affected by the polymer pKa. We suggest that the pH sensitive polymersomes investigated in this work disassembled upon exposure to the endosomal pH, i.e. the intermolecular associations of the polymer chains to form vesicles were disrupted by protonation of the imidazole blocks, which in turn caused the vesicles to dissociate into free tri-block copolymer chains. At the same time, dsDNA entrapped in the polymersomes was able to diffuse away. However, in the presence of the now-charged polyimidazole blocks, ds-DNA would have associated strongly with the copolymers by electrostatic interactions. This hypothesis is supported by our earlier observation from electrophoresis assays showing that the ds-DNA sequences remained tightly complexed to


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the polymer chains at pH 5.0. However, in the cytosol at pH 7.4 these individual oligonucleotide/copolymer complexes were likely to dissociate again (as suggested also by the earlier gel shift assays), allowing the released oligonucleotide to perform its biological role. The capacity of the nanocarrier to deliver and release ds-siRNA sequences in the active form in the cytosol through an endosomal escape mechanism was investigated with ‘knockdown’ studies using luciferase and Hsp90 silencing ds-siRNA sequences loaded in the targeted polymersomes. The active targeting capacity, the endosomal escape activity and the pH controlled release of the polymeric vesicles were shown to participate together to yield a significant silencing effect toward both the luciferase and the Hsp90 expression. No silencing effect was observed when cells were incubated with free unformulated luciferase silencing ds-siRNA showing that the nanocarrier formulation was essential to guarantee the access of the polyanionic macromolecules to the cytosol. The decrease of bioluminescence cannot be ascribed to an effect of the nanocarrier on the cell biological functions since the bioluminescence was not altered when cells were incubated solely with the void targeted polymeric vesicles. After the preliminary silencing results with luciferase, the polymeric vesicles were exploited to deliver a ds-siRNA sequence with therapeutic relevance. The oligonucleotide chosen was a sequence aimed to silence a heat-shock protein, Hsp90, commonly overexpressed in a number of human cancers.61 Hsp90 is a chaperone protein that participates in the folding of several "client" proteins in melanoma cells.62 Most heat-shock proteins (Hsps) are constitutively expressed to guide the appropriate folding, intracellular disposition and proteolytic turnover of many key regulators of cell growth and survival. Their levels of intracellular expression increase under protein-denaturing stress such as temperature change, as an evolutionarily response to enhance


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cell survival. The expression of the Hsp90 protein has been reported to be increased in many cancer cells and this has been associated with disease progression.63 In addition, Hsp90 takes part in processes relating to the folding of other overexpressed or mutated proteins in cancer cells that are also involved in cancer progression and resistance to therapy.64 For these reasons, inhibition of Hsp90 might result in a significant decrease in the function of proteins responsible for drug resistance, which in turn can restore the sensitivity towards anticancer drugs. We thus considered siRNA to be an attractive alternative to small molecule inhibitors to reduce the expression of Hsp90, and which might be delivered via polymersomes as the first steps towards combined biomolecular gene knockdown and pro-apoptotic small molecule anticancer agents.61 In our assays, a reduction of Hsp90 expression in B16-F10 cells after incubation with folate targeted polymersomes loaded with Hsp90 silencing ds-siRNA was shown, by Western blotting and quantification of protein expression. It is important to note that Hsp90 is a constitutively-expressed protein and thus the inhibition outcome was not affected by any potential bias deriving from tests using transfected cells such as the luciferase inhibition assays. The partial silencing of Hsp90 expression, which was also observed with the luciferase transfected cell model, can be attributed to a number of factors, each of which highlights a barrier to siRNA delivery in general. Sub-optimal delivery of siRNA into the cytosol may have occurred either through incomplete endosomal disruption or via incomplete or slow de-complexation of the ds-siRNA from the PEG-pImHeMA-pGMA chains, which has been reported previously for polyplexes.60 In addition, the apparent trafficking of ds-DNA sequences to the nucleus suggests a very tight complexation of the analogous ds-siRNA sequences with the polymers. This in turn would not have allowed sufficient oligonucleotide to escape to reach the RISC complex in the cytosol and enable the subsequent siRNA silencing to


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take place. The differences in knockdown compared to the commercial lipid-based transfection reagent may well have been due to this tight complexation between the tri-block copolymer and RNA compared to cationic lipid-RNA complexes. The lower biological response observed with the polymersomes compared to DharmaFECT® 1 may also be ascribed to the different cell-up take mechanisms of the two formulations. Indeed, the cell internalization of DharmaFECT® 1 is expected to occur by non-specific cell interaction while the targeted polymersomes are internalized by selective active mechanism. Therefore, the amount and rate of ds-siRNA delivery can be significantly different for the two formulations. Nevertheless, the partial inhibition of Hsp90 expression achieved with the pH responsive vesicular systems is promising for further investigations, as the polymersomes were stable, well-tolerated by a number of cell lines, and able to be functionalized and targeted by accessible synthetic chemistries. A higher stability of interaction between polymer and RNA might be more favorable in vivo, owing to the longer transport times and extended distribution. We are currently embarking on experiments to test these hypotheses in a range of more complex biorelevant cell models and in vivo.

CONCLUSIONS These studies showed that pH responsive triblock copolymers which self-assemble into nanosized vesicular systems were able to interact efficiently with oligonucleotide sequences that assisted encapsulation of the therapeutic biomolecules in colloidal vesicles. The loading of the oligonucleotides took place beneath the vesicle corona as demonstrated by the neutral zeta potential of the ds-DNA and ds-siRNA loaded polymersomes. Furthermore the oligonucleotides were tightly associated to the carrier, which enabled their efficient delivery to cancer cells according to a folate mediated active targeting process. The pH responsive character of the


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polymer forming the vesicles was shown to endow membrane disrupting properties at endosomal pH while retaining excellent blood cell compatibility at plasma and cytosolic pH. The selective delivery of labelled oligonucleotides was confirmed by flow cytometry and confocal microscopy; the latter also confirmed the endosomal escape of the oligonucleotide and their trafficking to the cytosol and migration to the nucleus at some extent. The folate targeted polymeric vesicles were loaded with biologically functional ds-siRNA sequences. Target protein expression inhibition was shown to take place following intracellular delivery and release of ds-siRNA in the active form to cancer cells. The tri-block materials used in this work exhibit a number of useful properties that are desirable for pharmaceutical applications. The ability to encapsulate macromolecules such as DNA and siRNA is a clear advantage for biomedical applications and enhanced therapeutic selectivity, since many new therapeutic molecules (namely proteins and siRNA) are biological in nature and are still in search for an efficient delivery system to be used in practice. In addition, the ability to fine tune the vesicle response across physiological pH ranges through functional monomer iteration may allow control over tissue localization and timely intracellular release as a consequence of the instructions ‘coded’ into the nanovehicles. An additional, non-trivial, advantage of using polymer based vesicular nanocarriers arises from their flexibility to be used for different cancer types by simply replacing the targeting agent and the siRNAs to silence a variety of target proteins. Nevertheless, future developments of these classes of materials will be needed to address important translational objectives such as good biodegradability, prevention of immune activation, accessibility from sustainable chemical or biological feedstocks as well as overall enhanced gene silencing activity. Studies to address these factors are ongoing in our laboratories.


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

AUTHOR INFORMATION *Corresponding author: Stefano Salmaso Department of Pharmaceutical and Pharmacological Sciences University of Padova Via F. Marzolo 5 35131 Padova – Italy Tel: +390498271602 Fax: +390498275366 e-mail: [email protected]

ACKNOWLEDGEMENTS We thank the Italian CNR (grant CUP: B51J09000200005), the UK Engineering and Physical Sciences Research Council (EPSRC: grants EP/H006915/1 and EP/H005625/1 Leadership Fellowship to CA), the Spanish MICINN (CTQ2010-18195/BQU, EUI2008-03905) and the European Commission that, through the ERANET NanoSci-E+ program, financially supported this project (acronym INANONAK).


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(60) Zhang, C.; Gao, S.; Jiang, W.; Lin, S.; Du, F.; Li, Z.; Huang, W., Biomaterials 2010, 31, 6075-6086. (61) Whitesell, L.; Lindquist, S. L., Nat. Rev. Cancer 2005, 5, 761-772. (62) Samadi, A. K.; Zhang, X.; Mukerji, R.; Donnelly, A. C.; Blagg, B. S.; Cohen, M. S., Cancer Lett. 2011, 312, 158-167. (63) Kamal, A.; Thao, L.; Sensintaffar, J.; Zhang, L.; Boehm, M. F.; Fritz, L. C.; Burrows, F. J., Nature 2003, 425, 407-410. (64) Wei, L.; Liu, T. T.; Wang, H. H.; Hong, H. M.; Yu, A. L.; Feng, H. P.; Chang, W. W., Breast Cancer Res. 2011, 13, R101.


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Biomacromolecules

“For Table of Contents Use Only"

Triblock copolymer nanovesicles for pH-responsive targeted delivery and controlled release of siRNA to cancer cells.

Elena Gallon,a Teresa Matini,b Luana Sasso,b Giuseppe Mantovani,b Ana Armiñan de Benito,c Joaquin Sanchis,c Paolo Caliceti,a Cameron Alexander,a* Maria J. Vicentc*and Stefano Salmasoa*


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Triblock Copolymer Nanovesicles for pH-Responsive Targeted

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