Demonstrating the importance of polymer-conjugate conformation in solution on its therapeutic output: Diethylstilbestrol (DES)-polyacetals as prostate cancer treatment

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Journal of Controlled Release 159 (2012) 290–301

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Demonstrating the importance of polymer-conjugate conformation in solution on its therapeutic output: Diethylstilbestrol (DES)-polyacetals as prostate cancer treatment Vanessa Giménez a, Craig James b, Ana Armiñán a, Ralf Schweins c, Alison Paul b, María J. Vicent a,⁎ a b c

Polymer Therapeutics Lab., Centro de Investigación Príncipe Felipe (CIPF), Av. Autopista del Saler 16, 46012 Valencia, Spain School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK Institute Laue-Langevin, BP 156, 6, rue Jules Horowitz, 38042 Grenoble Cedex 9, France

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Article history: Received 4 September 2011 Accepted 24 December 2011 Available online 31 December 2011 Keywords: Polymer therapeutics Polyacetals pH-dependent degradation Solution conformation SANS Prostate cancer

a b s t r a c t The design of improved polymeric carriers to be used in the next generation of polymer therapeutics is an ongoing challenge. Biodegradable systems present potential advantages regarding safety benefit apart from the possibility to use higher molecular weight (Mw) carriers allowing PK optimization, by exploiting the enhanced permeability and retention (EPR)-mediated tumor targeting. Within this context, we previously designed pH-responsive polyacetalic systems, tert-polymers, where a drug with the adequate diolfunctionality was incorporated within the polymer mainchain. The synthetic, non-steroidal estrogen, diethylstilboestrol (DES) clinically used for the treatment of advanced prostate cancer was chosen as drug. In order to improve the properties of this tert-polymer, novel polyacetalic systems as block-co-polymers, with more defined structure have been obtained. This second generation polyacetals allowed higher drug capacity than the tert-polymer, a biphasic DES release profile at acidic pH and due to its controlled amphiphilic character readily formed micelle-like structures in solution. These features result in an enhancement of conjugate therapeutic value in selected prostate cancer cell models. Exhaustive physico-chemical characterization focusing on nanoconjugate solution behavior and using advanced techniques, such as, pulsed-gradient spin-echo NMR (PGSE-NMR) and small-angle neutron scattering (SANS), has been carried out in order to demonstrate this hypothesis. Clear evidence of significantly different conformation in solution has been obtained for both polyacetals. These results demonstrate that an adequate control on molecular or supramolecular conformation in solution with polymer therapeutics is crucial in order to achieve the desired therapeutic output. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The development of better polymeric carriers is an ongoing challenge to achieve the next generation of polymer therapeutics [1-3]. Biopersistent carriers as polyethylenglycol (PEG) or N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers can present disadvantages if chronic parenteral administration and/or high doses are required as there is the potential to generate ‘lysosomal storage disease’ syndrome. Preclinical evidence of intracellular vacuolation with certain PEG-protein conjugates is raising awareness of the potential advantage of biodegradable polymers regarding safety benefit apart from the possibility to use higher molecular weight (Mw) carriers allowing PK optimization, by enhancing the enhanced permeability and retention (EPR)-mediated tumor targeting [2,4,5]. Therefore, biodegradable polymers such as dextrins [6,7], polypeptides [4,8,9], polyesters [10,11] or polyacetals [12,13] could be

⁎ Corresponding author. Tel.: + 34 963289681; fax: + 34 963289701. E-mail address: [email protected] (M.J. Vicent). 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.12.035

considered as promising candidates to be used as carriers for targeted drug delivery. To allow use of polymers of higher Mw, a family of hydrolytically labile water-soluble polyacetals was developed [13]. These can be functionalized to allow side-chain conjugation to a drug payload such as doxorubicin (Dox) [12]. These polyacetals show a clear pHdependent degradation being relatively stable at pH 7.4 but degrade significantly faster at the acidic pH that is encountered in endosomes and lysosomes. In vitro and in vivo studies confirmed that the polyacetals are not toxic, they are not taken up extensively by the liver or spleen, and are also long circulating [13]. Moreover, the polyacetal– Dox conjugate (Mw 86 kDa) displayed significantly prolonged plasma circulation time and enhanced tumor accumulation compared to the HPMA copolymer-Dox conjugate (CF28068, known as PK1, Mw 30 kDa) in Phase II clinical trials [12,14]. Polyacetals can be prepared by a mild polymerization method involving the reaction of diols with divinyl ethers [15]. To move a step further on this design we synthesized polyacetals incorporating a drug with bis-hydroxyl functionality into the polymer backbone [16]. Degradation of the polymer backbone in the acidic environment of the lysosome or the extracellular fluid of some tumors would then

trigger drug release eliminating the need for a biodegradable linker. For this purpose, we used the tert-polymerization process developed for the synthesis of the functionalized polyacetals [13] in combination with the model drug diethylstilbestrol (DES) [16]. DES is a synthetic non-steroidal estrogen and its administration was a classic form of androgen deprivation therapy (ADT), standard approach to the treatment of advanced prostate cancer for more than 50 years. Its use, however, has been severely limited by a poor water solubility and wide ranging dose-related toxicities, mainly cardiovascular side effects and in particular thromboembolic events [17-20]. DES can be considered as an ‘old’ treatment, however, is taken renewed consideration as very recently has been demonstrated that low-dose DES is safe and effective in castrate-resistant prostate cancer (CRPC) patients when used before the initiation with chemotherapy [19]. Also, a combination of DES to chemotherapeutics such as docetaxel was found to produce a significant level of antitumor activity in patients with metastatic, androgen independent prostate cancer (AIPC) [21,22]. It is hypothesized that, apart from clearly reducing DES toxicity by means of the EPR-mediated tumor targeting [1,23], the conjugation of DES to polymeric carriers would more easily allow a lowdose clinical regime as a controlled release of the drug could be achieved for a prolonged period of time. Also, polymer multivalency would allow the synthesis of polymer-based combination conjugates that could better exploit the synergism observed already with, i.e. docetaxel [21,24]. Our previous research with DESpolyacetals already demonstrated that DES solubility can be greatly enhanced upon polymerization. And more interestingly, the conjugates underwent degradation that was clearly pH-dependent, with greater DES release at acidic pHs. Additionally, the active isomerism of the estrogen was maintained (trans-DES) [25] and the conjugates displayed enhanced in vitro cytotoxicity compared to free DES. These tert-DES polyacetals could therefore be defined as the first water-soluble anticancer polymeric drugs designed for acidic pH-triggered release where the drug is incorporated into the polymer mainchain [16]. Ratifying the utility of this synthetic strategy, another recent example has been reported using curcumin as a diolfunctionalized anticancer drug. The polyacetal-based polycurcumins showed a clear antitumor effect in vitro and in vivo in ovarian cancer models [26]. The first synthesized tert-polymer had a drug content of ~4 wt.% and a polydispersity (Mw/Mn) around 1.8. The initial aim of the current study was to synthesize a second generation of DES-based polyacetals with improved properties, such as narrower Mw distributions and higher drug loading, and more importantly to study with these model systems, if slight structural modifications could significantly influence conjugate therapeutic output. These second generation polyacetals were obtained using a block-co-polymer methodology. Tert-DES and block-DES were then tested in selected prostate cancer cell models. In order to explain the differences encountered once biologically evaluated, an exhaustive characterization of the conformation in solution for both polyacetalic systems was carried out by means of different techniques, such as: transmission electron microscopy (TEM), dynamic light scattering (DLS), pulsed-gradient spinecho NMR (PGSE-NMR) [27] and small-angle neutron scattering (SANS) [28] which have recently been used to good effect for understanding the solution conformation of polymer-conjugates [29-32]. 2. Materials and methods 2.1. Materials and instruments Tri(ethylene glycol) divinyl ether (TEGDVE), 2-amino-1,3-propanediol (Serinol), Fluorenylmethyloxycarbonyl chloride (Fmoc-Cl), Triethylamine and N,N-dimethylformamide (DMF) were from Fluka Chemika (Masserschmittstr, D). Dioxane, tetrahydrofuran anhydrous (THF) and toluene anhydrous were supplied from Aldrich (Dorset,

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UK). Before use, the THF was distilled from sodiumbenzophenone. Poly (ethylene glycol) (PEG), p-toluenesulfonic acid monohydrate (p-TSA), diethylstilboestrol (DES) were obtained from Aldrich and dried in a vacuum oven at 80 °C before use. Deuterated chloroformd1, DMSO-d6 and D2O were purchased from Deutero GmbH. DMSOd6 was dried and stored over molecular sieves (4 Å). Deuterated methanol MeOD (>99%-d) was from Sigma-Aldrich (UK), and used as received. 2.2. Characterization 1

H- and 13C-NMR spectra were obtained at 300 MHz using a FTspectrometer from Bruker and analyzed using the Topspin software. Pulsed Gradient Spin-Echo (PGSE) NMR experiments were performed on a Bruker AMX360 NMR spectrometer using a stimulated echosequence as described elsewhere [33]. The configuration uses a 5 mm diffusion probe (Cryomagnet Systems, Indianapolis) and a Bruker gradient spectroscopy accessory unit. The polymer selfdiffusion coefficients Ds were obtained for solutions at 25 °C using methodology and analysis previously described [34]. For size exclusion chromatography (SEC) measurements the polymers were dried at 40 °C overnight in a vacuum oven and subsequently characterized by SEC at 25 °C with THF as the eluent at a flow rate of 0.8 mL min − 1. Prior to injection the samples were filtered (0.22 μm nylon membrane) and sonicated for 5 min. The SEC equipment consisted of a Waters 717 plus autosampler, a TSP Spectra Series P 100 pump triSEC-Viscotek, array, TDA™ 302 series system, including two Waters Styragel 7.8 × 300 mm Columns (HR3 and HR4) for THF/DMF and a Viscotek TDA 302 triple detector Array with refractive index (RI), Small Angle Light Scattering, Right Angle Light Scattering, viscosimeter and a model 2501 UV as detector. Mw/Mn and Mw of the polyacetals were determined using the software OmniSec 4.2. Calibration was achieved with well defined Poly(methyl methacrylate) (PMMA)/THF standards (PolyCAL PMMA 65KDa Standard, dn/dc = 0.0854 mL/g; Mn = 61304 Mw = 64368 IV = 0.228), provided by Polymer Standards Service (PSS)/Mainz Germany. MTT assay measurements were performed using a Victor 2 Wallac 1420 Multilabel HTS Counter Perkin Elmer plate reader (Northwolk, CT, USA). Live cell confocal fluorescence microscopy studies were carried out at the confocal microscopy service at CIPF (Valencia, Spain) and were performed using a Leica confocal microscope from Leica Microsystems GmbH (Wetzlar, D) equipped with a l-blue 63 oil immersion objective and handled with a TCS SP2 system, equipped with an acoustic optical beam splitter (AOBS). Excitation was with an argon laser (548, 476, 488, 496 and 514 nm) and blue diode (405 nm). Images were captured at an 8-bit gray scale and processed with LCS software Version 2.5.1347 (Leica, Germany) containing multicolor, macro and 3D components. DES-Polyacetal (tert-DES) 1 was synthesized by tertpolymerization of vinyl ethers and alcohols in THF by optimization of the previously reported protocol [16]. Briefly, PEG (2 g, Mw = 4.000 g/mol, 0.5 mmol), p-TSA (0.003 g, 0.015 mmol) and DES (0.134 g, 0.5 mmol) (all previously dried for 24 h in a vacuum oven) were added to THF (6 mL, distilled over sodium) and stirred until dissolved. Next, TEGDVE (0.2 mL 1.07 mmol) was slowly added to the solution using a syringe to preserve anhydrous conditions. The reaction was stirred vigorously for 3 h in the dark at RT. Triethylamine was then added to neutralize the p-TSA catalyst (until pH 8) and then followed by rapid stirring for 30 min. The solution was then poured into a rapidly stirring hexane/diethyl ether mixture (4:1, 100 mL) cooled over an ice bath, and stirred for a further 30 min to fully precipitate polyacetal 1. The product was isolated as a white powder by filtration and further dried in vacuum at RT. To ensure complete removal of p-TSA an extraction with CHCl3 and saturated NaHCO3 solution was carried out and afterwards the organic phases

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were combined, washed with saturated NaCl and dried over Na2SO4. The solvent was removed under vacuum and the residue was redissolved in MilliQ water and lyophilized to yield tert-DES as a white solid. Yield: 87%. Tert-DES 1: 1H NMR (CDCl3, 300 MHz): 0.77 (t, J = 7.5 Hz, DES CH3), 1.33 (d, J = 3.9 Hz, PEG-acetal CH3), 1.53 (d, J = 5.4 Hz, DES-acetal CH3), 2.13 (dd, J = 7.5 Hz, J = 15.3 Hz, DES CH2), 3.59 (m, PEG CH2), 4.80 (q, J = 3.9 Hz, PEG-acetal CH), 5.46 (q, J = 5.4 Hz, DES-acetal CH), 7.0–7.2 (m, DES ArH). 13C NMR (CDCl3, 75 MHz): 13.40, 19.55, 20.16, 64.09, 64.59, 70.55, 99.64, 99.82, 116.82, 129.66, 130.73, 136.20, 138.69, 151.74, 155.24. DES-co-polyacetal-b-PEG-co-Polyacetal (Block-DES) 2. PEG (2 g, Mw = 4000 g/mol, 0.5 mmol) and p-TSA (0.003 g, 0.015 mmol) were added to an oven dried flask and then dissolved in THF (4 mL, anhydrous). Next, TEGDVE (0.22 mL, 0.5 mmol) was added and the mixture was stirred vigorously for 3 h in the dark at RT. Afterwards, DES (0.134 g, 0.5 mmol) was slowly added to the reaction using a syringe to preserve anhydrous conditions and stirred for a further 3 h in the dark at RT. The reaction was stopped by addition of triethylamine which was added to pH 8 to neutralize the p-TSA catalyst. After stirring for 30 min the solution was poured into a cold hexane: diethyl ether mixture (4:1, 100 mL) to precipitate the polyacetal 2 whilst stirring. After an additional 30 min the polymer was collected and placed into a fresh solution of hexane: diethyl ether (4:1) and stirred for a further 30 min to achieve an exhaustive wash. The polyacetal was again collected and then dried under vacuum for 24 h to obtain the polymer as a white solid (1.6 g). Yield: 85%. Block-DES 2: 1H NMR (CDCl3, 300 MHz): 0.77 (t, J = 7.5 Hz, DES CH3), 1.25 (d, J = 3.8 Hz, PEG-acetal CH3), 1.54 (d, J = 5.4 Hz, DES-acetal CH3), 2.15 (dd, J = 7.6 Hz, J = 15.4 Hz, DES CH2), 3.4–3.9 (m, PEG CH2), 4.78 (q, J = 4.0 Hz, PEG-acetal CH), 5.47 (q, J = 5.5 Hz, DES-acetal CH), 7.0– 7.2 (m, DES ArH). 13C NMR (CDCl3, 75 MHz): 13.44, 19.53, 20.15, 28.51, 64.04, 64.55, 70.52, 99.59, 99.78, 114.42, 114.98, 116.78, 129.64, 130.69, 134.06, 136.35, 138.35, 138.93, 154.97, 155.16, 155.20. 2.3. Synthesis of Fmoc-N-protected serinol The method to synthesize hydroximethylaziridines was adapted from literature [12]. The synthesis was carried out by condensation between the acyl group in Fluorenylmethyloxycarbonyl (Fmoc) and the amine group in Serinol to form an amide bond by elimination of HCl. Serinol (1.0 g, 11 mmol) was dissolved in a 10% Na2CO3 solution (26.5 mL). Then, dioxane (15 mL) was added and the mixture was stirred in an ice water bath. Fmoc-Cl (2.86 g, 11 mmol) was carefully added to the mixture and then stirred for 4 h at 4 °C (after 2 h, 15 mL dioxane were added to decrease viscosity). The mixture was then stirred overnight at RT (ca. 16 h). After this, distilled water (100 mL) was added and the product extracted with ethyl acetate (2 × 100 mL). The organic phases were combined and dried over Na2SO4. After filtration the solvent was removed under vacuum to obtain a white solid. Then the solid was redissolved in 40 mL dioxane and precipitated over 150 mL hexane. Yield: 84%. Fmoc-N-protected serinol: 1H-NMR (δH, 300 MHz, CDCl3) 3.76–3.36 (5 H, m, ―CH2―OH overlap with ―CHN―), 4.17–4.13 (1 H, t, J = 6.6 Hz, ArCH-Ar), 4.39 – 4,37 (2 H, d, J = 6.3 Hz, O―CH2―), 7.28–7.22 (2H, t, d, Jm = 1.2, Jo = 7.5 Hz, Ar), 7.37–7.36p (2 H, t, d, Jm = 1.2, Jo = 7.5 Hz, Ar), 7.54–7.52 (2 H, d, Jo = 7.5 Hz, Ar), 7.71–7.69 (2 H, d, Jo = 7.5 Hz, Ar). 2.4. Fmoc-protected DES-Serinol-polyacetals 2.4.1. tert-DES-FmocSerinol Firstly, the reagents, PEG4000, p-TSA and Fmoc-Serinol were dried separately at 80 °C under high vacuum overnight. Then, PEG (1 g, Mw = 4.000 g/mol, 0.25 mmol), p-TSA (0.0002 g, 0.009 mmol), Fmoc-Serinol (0.053 g, 0.25 mmol) and DES (0.062 g, 0.25 mmol)

were transferred into a 2-neckround bottomed flask and purged with nitrogen. The reagents were then dissolved in THF (4 mL, distilled). The reaction mixture was stirred for 20 min while purging with nitrogen. Following this, TEGDVE (0.33 mL, 0.75 mmol) was added directly to the reaction and after stirring for 3 h, triethylamine (0.2 mL) was added until pH 8 and the reaction was stirred for a further 45 min. Next, the reaction mixture was precipitated into a cold hexane: diethyl ether mixture (4:1, 100 mL) and stirred for 15 min. Finally, the resulting polymer was filtered to obtain the desired product at 77% yield. tert-DES-FmocSerinol : 1H NMR (CDCl3, 300 MHz): 0.77 (t, J = 7.5 Hz, DES CH3), 1.33 (d, J = 3.9 Hz, PEG-acetal CH3), 1.50–1.52 (d, J = 5.4 Hz, DES-acetal CH3), 2.13–2.3 (dd, J = 7.5 Hz, J = 15.3 Hz, DES CH2), 3.4–3.6 (m, PEG CH2, m, Serinol-CH2), 4.1–4.2 (t, Fmoc Ar CH-CH2-), 4.3–4.4 (t, J = 3.1 Hz, Fmoc Ar-CH-CH2-) 4.80 (q, J = 3.9 Hz, PEG-acetal CH), 5.46 (q, J = 5.4 Hz, DES-acetal CH), 7.0–7.2 (m, DES ArH) 7.3–7.8 (m, Fmoc ArH). 13 C NMR (CDCl3, 75 MHz): 13.42, 19.59, 20.43, 63.97, 64.39, 70.45, 100.21, 100.43, 114.82, 116.82, 129.46, 130.70, 136.70, 138.76, 151.93, 155.14, 51.3, 47.34, 64.2, 68.76, 126.7, 127.7, 128.7, 156.5. 2.4.2. Block-DES-FmocSerinol PEG4000 (0.61 g, Mw = 4.000 g/mol, 0.15 mmol), p-TSA (0.0001 g, 0.004 mmol) and Fmoc-Serinol (0.064 g, 0.204 mmol) were dried in separate vessels at 80 °C in vacuo overnight. PEG and p-TSA were weighed directly into a 2- neck flask and purged with nitrogen. THF was added to the flask and the mixture was heated to improve the solubility of the reactants. After cooling down to RT, TEGDVE (0.375 mL, 0.85 mmol) was added directly to the reaction and after 3 h stirring, p-TSA dissolved in 1 mL THF and TEGDVE dissolved in 8 mL THF were added. After 30 min, DES (0.062 g, 0.25 mmol) and Fmoc-Serinol (0.064 g, 0.204 mmol) were dissolved together in THF, added to the reaction and left stirring for 3 h. Then, triethylamine was added up to pH ~ 8. Finally, the reaction mixture was precipitated over a cold hexane: diethyl ether mixture (4:1) for 15 min and after filtration polyacetal 5 obtained in a 34% yield. Block-DES-FmocSerinol: 1 H NMR (CDCl3, 300 MHz): 0.7–0.8 (t, J = 7.4 Hz, DES CH3), 1.2–1.3 (d, J = 3.8 Hz, PEG-acetal CH3), 1.4–1.5 (d, J = 5.5 Hz, DES-acetal CH3), 2.1–2.2 dd, J = 7.6 Hz, J = 15.4 Hz, DES CH2), 3.5–3.9 (m, PEG CH2, m, Serinol-CH2), 4.1–4.2 (t, Fmoc Ar CH-CH2-), 4.3–4.4 (t, J = 3.1 Hz, Fmoc Ar-CH-CH2-) 4.7–4.8(q, J = 3.9 Hz, PEG-acetal CH), 5.4–5.5 (q, J = 5.4 Hz, DES-acetal CH ), 7.0–7.2 (m, DES ArH) 7.3–7.9 (m, Fmoc ArH). 13C NMR (CDCl3, 75 MHz): 13.34, 19.54, 20.16, 28.51, 64.04, 64.55, 70.52, 99.61, 99.79, 114.52, 115.12, 116.69, 116.78, 129.74, 130.68, 134.12, 136.25, 138.45, 138.95, 154.68, 155.34, 155.19, 51.32, 47.03, 64.24, 68.3, 126.57, 127.31, 129.3, 156.52. 2.5. Polyacetal deprotection Tert-DES-Ser 3 and Block-DES-Ser 4 were synthesized by removal of Fmoc groups. The polymers were individually dissolved in a 20% piperidine/acetonitrile solution (10 mL) and stirred for 1 h. The reaction was monitored by TLC (100% ethyl acetate, Rf = 0.7). The reaction mixture was washed with hexane (3 × 15 mL) and the acetonitrile removed under vacuum at RT. Then, the residual solid was redissolved in 15 mL of hexane and stirred for 2 h and the polyacetals 3 and 4 obtained (90% yield). Tert-DES-Ser 3 : 1H NMR (CDCl3, 300 MHz): 0.79 (t, J = 7.3 Hz, DES CH3), 1.29 (d, J = 3.6 Hz, PEGacetal CH3), 1.48–1.51 (d, J = 4.8 Hz, DES-acetal CH3), 2.11–2.2 (dd, J = 7.6 Hz, J = 14.9 Hz, DES CH2), 3.58–3.91 (m, PEG CH2), 4.1–4.2 (t, Fmoc Ar CH-CH2-), 4.3–4.4 (t, J = 3.1 Hz, Fmoc Ar-CH-CH2-) 4.81 (q, J = 3.9 Hz, PEG-acetal CH), 5.45 (q, J = 5.3 Hz, DES-acetal CH), 6.9– 7.1 (m, DES ArH). 13C NMR (CDCl3, 75 MHz): 13.43, 19.58, 20.16, 28.62, 47.56, 64.11, 65.01, 67.51, 70.56, 99.66, 99.82, 114.41, 114, 89, 116, 57, 116.82, 128.92, 130.73, 135.60, 138.71, 138, 89, 151.72, 156.24, 155.81.

Block-DES-Ser 4: 1H NMR (300 MHz, CDCl3): 0.7–0.8 (t, J = 7.4 Hz DES CH3), 1.2–1.3 (d, J = 3.7 Hz, PEG-acetal CH3), 1.5–1.6 (d, J = 5.1 Hz DES-acetal CH3), 2.1–2.2 (dd, J = 7.5 Hz, DES CH2), 3.49–3.9 (m, PEG CH2) 4.1–4.2 (t, Fmoc Ar CH-CH2-), 4.3–4.4 (t, J = 3.1 Hz, Fmoc Ar-CH-CH2-), 4.7–4.8 (q, J = 4.1 Hz PEG acetal CH), 5.4–5.5 (q, J = 5.5 Hz DES-acetal CH), 7.0–7.2 (m, DES ArH). 13C NMR (CDCl3, 75 MHz): 13.45, 19.8, 19.98, 28.58, 47.5, 64.21, 64.56, 67.1, 71.42, 99.69, 99.79, 114.52,114.88, 116.53, 116.79, 129.74, 130.74, 134.45, 136.66, 138.95, 138.99, 154.77, 155.15, 155.21. 2.6. Fluorescently labeled polyacetals tert-DES-Ser-OG 5 and Block-DES-Ser-OG 6 A fluorescence probe, NHS activated Oregon Green (OG) was conjugated to polyacetals 3 and 4 through free amino groups, in order to study the cellular uptake and trafficking in selected prostate cancer cell models. Polymers 3 (0.300 g, 0.030 mmol) and 4 (0.300 g, 0.022 mmol) were dissolved in anhydrous THF and DIEA was then added until pH 9. NHS activated OG (Mw = 0.0002 g, 0.0008 mmol) dissolved in CH2Cl2 was then added to the reaction mixture and left to react for 16 h. The reaction was monitor by TLC (Ethyl acetate: hexane, 1:1, Rf = 0.5). After solvent removal under vacuum at RT, the residual product was redissolved in methanol and loaded in SEC column (sephadex LH20) eluted with methanol, collecting fractions of 1 mL. Each fraction (2 μL) was taken and added to MeOH (498 μL), in order to measure the fluorescence and to identify the fractions containing the labeled polyacetals and also to quantify the amount of conjugated probe. OG loading ranged from 0.5 to 0.8 mol%. Determination of possible free OG in the conjugate was determined by HPLC (always free drug content b0.5 wt.% of total drug). 2.7. Investigation of pH-dependent degradation Polyacetals (8 mg/mL) were incubated at 37 °C in phosphate buffer solution (PBS) at pH 5.5, 6.5 and 7.4 for 20 days. 100 μL for HPLC and 50 μL for GPC analysis of the sample solutions were taken at various time points 0, 15, 30 min and 2, 8 and 24 h and every 24 h up to achieve complete degradation. Samples were frozen with liquid nitrogen and stored at −80 °C until analyzed. Prior to analysis, the pH of acidic samples was neutralized with ammonium formate buffer (0.1 M, 100 μL for pH 5.5 and 50 μL for 6.5), in order to stop any further degradation, to normalized concentrations 100 μL PBS was added to the samples of pH 7.4 and 50 μL to the sample of pH 6.5. Next, the samples were directly analyzed either by GPC (%Mw Loss, PBS as mobile Phase, flow 0.8 mL/min) and by RP-HPLC, using a C18 LiChroSpher 100 column (5 μm), with the UV detector settled at λ = 280 nm with a flow rate of 1 mL/min. The eluent A was H2O and eluent B was MeCN. Oestradiol was used as HPLC internal reference standard; 100 μL of a 10 μg/mL stock solution was added to each sample. The elution was performed by the following gradient: from 30% B to 90% B over 25 min, 3 min isocratic, then from 90% B to 30% B over 3 min and keeping these conditions for 4 min (trans-DES retention time (tr) 11 min, cis-isomer tr 13 min, oestradiol tr 10 min). A calibration curve of DES was used to quantify the total DES release from the conjugates by HPLC.

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2.9. Cell cultures Biological studies were carried out in two different human prostate cancer cell lines, PC3 hormone independent and LNCaP hormone dependent cell lines. PC3 were grown in F12 medium with 5.0 mM Lglutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS) and LNCaP in RPMI medium supplemented with 10% (v/v) FBS. Cells were maintained at 37 °C in an atmosphere of 5% carbon dioxide and 95% air and underwent passage twice weekly. NaDES used in cell culture experiments was obtained as previously reported [16]. 2.10. Confocal fluorescence microscopy: live cell imaging PC3 and LNCaP cells were seeded at a density of 3.2 × 10 4 cell/mL and 5 × 10 4 cell/mL, respectively, on glass bottom culture dishes (10 cm 2 Petri plate) and allowed to seed for 24 h. Then, OG-labeled conjugates were added (0.4 and 0.6 mg/mL). Pulse and chase experiments were performed at various time points up to 5 h incubation at 37 °C then the medium was removed and the cells were washed twice with PBS supplemented with 10% (v/v) of fetal bovine serum (FBS, 3 mL, 37 °C) and then the glass removed and placed on the microscope chamber. In order to capture the images, in selected samples the lysosomal marker Dextran-Texas red was also used to identify possible co-localization and therefore establish an endocytic pathway. A volume of 5 μL of a Dextran-Texas Red solution (1 mg/mL) was added and after 1 h incubation, the medium was replaced and the cells incubated for further 5 h. 2.11. Cytotoxicity of free DES and DES-polyacetals The cytotoxicity of the free DES and the polyacetals synthesized was evaluated using an MTT cell viability assay after 72 h incubation against PC3 and LNCaP cells [35]. Cell lines were seeded in sterile 96-well microtitre plates at a seeding density of 3.2 × 10 4 cell/mL and 5 × 10 4 cell/mL, respectively. Plates were incubated for 48 h and the compounds were then added to give a final concentration in the range of 0–1 mg/mL drug-equiv. After 67 h incubation, MTT (20 μL of a 5 mg/mL solution in PBS) was added to each well, and the cells were incubated for further 5 h. The medium was removed and the precipitated formazan crystals were dissolved in optical grade DMSO (100 μL), then 30 min later, the plates were analyzed by spectrophotometry at 550 nm using a microtitre plate reader. The measurements were taken in a Victor 2 Wallac 1420 Multilabel HTS Counter Perkin Elmer (Northwolk, CT, EEUU) unit. Cell viability was expressed as a percentage of the viability of untreated control cells. 2.12. Dynamic light scattering (DLS) studies DLS measurements were performed at 25 °C using a Malvern Zetasizer NanoZS instrument, equipped with a 532-nm laser at a fixed scattering angle of 90º. Polymer conjugate solutions (from 0.5 mg/mL to 3 mg/mL) were prepared using MilliQ H2O and phosphate buffer solution (PBS) at pH 7.4. The solutions were sonicated for 10 min and filtered through a 0.45 μm cellulose membrane filter before analysis. Micelle size distribution by volume (%) was measured (diameter, nm) for each conjugate (n ≥ 3).

2.8. Plasma stability 2.13. Transmission electron microscopy (TEM) Conjugates (8 mg/mL) were incubated at 37 °C in freshly extracted serum from Wistar rats for up to 24 h. At scheduled times, samples of 100 μL were collected; 10 μL of 100 μg/mL solution of oestradiol in MeOH, as internal standard, and 135 μL of MeCN were added to each sample in order to precipitate serum proteins. Following centrifugation (14000 rpm, 5 min), supernatants were analyzed by HPLC as reported above.

Polymer conjugate solutions, from 0.5 mg/mL to 3 mg/mL, were prepared using MilliQ H2O and PBS at pH 7.4. The solutions were sonicated for 10 min and filtered through a 0.45 μm cellulose membrane filter before analysis. The sample could be quickly prepared by the deposition of 1 μL of a dilute sample containing the polymer onto support films.

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2.14. Small-angle neutron scattering (SANS)

H

SANS measurements were performed at the ISIS spallation neutron source using the time-of-flight diffractometer, LOQ, in Oxfordshire (UK) and also performed in the reactor 57 MW HFR (High-Flux Reactor) at the Institute Max von Laue-Paul Langevin (ILL) in Grenoble, France. The conjugates were dissolved at 1 wt.% concentration of Polymer in D2O and MeOD, and scattering experiments were performed at 37 °C, conditions mimicking those experienced during cellular uptake. All experiments were performed in 2 mm quartz cells with typical measuring times of 1 h per sample.

O

O x H

+

O

O

O

O

TEGDVE

PEG

1) THF, p-TSA 30°C, 3h 2)TEGDVE 30min DES 3h 30°C, 3h

*

O

O3 O

O

O x

O

3

O O m

n-m

Scheme 1. Synthetic approach followed to obtain Block-DES-polyacetals 2.

2.15. Pulsed-Gradient Spin-Echo NMR (PGSE-NMR) The conjugates were dissolved at the same concentration used for SANS experiments, 1 wt.% concentration of polymer dissolved in D2O and MeOD. The self-diffusion coefficient Ds was extracted by fitting the integrals for a given peak to Eq. (1);
 13 2 0 2 3 2 2 3 30Δðδ þ σ Þ − 10δ þ 30σ δ þ 35σ δ þ 14δ 2 2 Aðδ; σ ; G; ΔÞ ¼ AO exp4−γ G @ Ds A5 30

ð1Þ

possible to introduce a drug within the polymer backbone, achieving the first water-soluble anticancer polymeric drug designed for acidic pH-triggered release using DES as drug [16]. Trying to move a step further in conjugate design and exploring how control on the polymerization procedure would influence biological output, we decided to design novel DES-polyacetals as block-co-polymers systems (block-DES) with amphiphilic character. It is important to note that in all studies tert-DES was used as reference polymer. 3.1. Synthesis and characterization of DES-polyacetals

where γ is the magnetogyric ratio and A is the signal amplitude in the absence (A0) or presence (A(δ,σ,G,Δ)) of the trapezoidal field gradient pulses of duration δ, ramp time σ, separation Δ and intensity G. A stretched exponential fit accounts for the moderate polydispersity of the sample. Typical values defining the gradient pulses were Δ = 200 ms, 500 μs b δ b 3 ms, and 0.5 b G b 3 T/m. 2.16. Statistical analysis All results are given as means ± SD (n ≥ 3). When only two groups were compared, the Student's t test for small sample size was used to estimate statistical significance. If more than two groups were compared evaluation of significance was performed using one wayAnalysis Of Variance (ANOVA) followed by Bonferroni post hoc test. Graph pad Instant software (Graph Pad Software Inc. CA, USA) was used. In all cases, statistical significance was set at p b 0.05. 3. Results and discussion Polyacetals are prepared by a mild polymerization method involving the reaction of diols with commercially available divinyl ethers [15]. We had already demonstrated that utilizing this chemistry is

A family of Tert-DES-polyacetals 1 (tert-DES) was synthesized following the tert-polymerization technique previously described and using 1H-NMR in order to confirm the presence of the drug in the polymer mainchain. 1H-NMR allowed quantification of DES loading as for the presence of two distinct sets of acetal peaks, which correspond to the two possible mainchain acetals; from PEG at 1.25–1.3(d) and 4.7–4.8(q)ppm and from DES at 1.5–1.6(d) and 5.4–5.5(q)ppm [16]. Water-soluble tert-DES polyacetals obtained had Mw range from 34000 g/mol to 38000 g/mol and with Mw/Mn from 1.5 to 1.7 as determined by size exclusion chromatography (GPC) (THF, 0.8 ml/min). DES total loading in the different watersoluble tert-polyacetals varied from 3 to 6 wt.% with a free drug content always b0.5 wt.% of total drug (Table 1, Scheme 1S, Fig. 1S). A DES loading greater than 6 wt.% yielded non-water soluble materials. In parallel, a family of amphiphilic block-co-polymers, Block-DESpolyacetals 2 (block-DES), was also synthesized. The synthetic strategy was based on the same polymerization technique described above but using a sequential approach (Scheme 1). The main synthetic differences for the tert- and block- polymers were based on the DES addition time point. When DES was added at the beginning of the reaction together with poly(ethylenglycol) (PEG) and tri(ethylene

Table 1 Physico-chemical characteristics of the polyacetals synthesized and used in this study. Conjugate

DES loadinga (wt.%)

Free DES contentb (wt.% of total drug)

Serinola,c loading (wt.%)

Oregon greend content (mol%)

Mwe (g/mol)

Mw/Mne

Tert-DES 1a Tert-DES 1b Tert-DES 1c Block-DES 2a Block-DES 2b Block-DES 2c Block-DES 2 d Tert-DES-Ser 3 Block-DES-Ser 4

2.8 ± 0.2 4.0 ± 0.2 5.7 ± 0.3 2.0 ± 0.1 4.3 ± 0.3 7.3 ± 0.3 9.5 ± 0.2 4.5 ± 0.3 4.7 ± 0.4

0.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.2 0.2 ± 0.1 0.1 ± 0.2 0.2 ± 0.3 0.3 ± 0.2 0.2 ± 0.1 0.4 ± 0.2

– – – – – – – 3.0 ± 0.3 3.0 ± 0.2

– – – – – – – 0.8 ± 0.2f 0.8 ± 0.2f

35.280 34.400 37.607 28.081 28.780 34.300 26.700 37.801 30.768

1.70 1.63 1.54 1.40 1.27 1.42 1.60 1.63 1.50

DES: Diethylstilbestrol, Ser: Serinol, OG: Oregon Green, Mw: Molecular weight, Mw/Mn: polydispersity index. a Determined by 1H NMR. b Determined by HPLC analysis. c Determined by nynhidrine assay. d Determined by Fluorimetry (Victor2 Wallac Station). e Determined by size exclusion chromatography (GPC, Viscotek TDA™). f Regarding block-DES-Ser-OG 5 and block-DES-Ser-OG 6, respectively.

k

O

O

k

o*O ** 3

p**

295

l m l

n

m

ñ

ñ m

n m

r**

l

l

k

k

O

O

O

n

k

3*

O

O

q*

k

x m

k

ml

p** o*

q*

n

r** ñ

Fig. 1. Example of 1H-NMR spectrum of block-DES 2 with assigned signals.

glycol) divinyl ether (TEGDVE) (in a one-pot polymerization approach) a tert-polymer was formed. In contrast, if DES incorporation was performed in a second step after PEG block formation an amphiphilic block-co-polymer could be achieved (See Supporting Information Scheme 1S, Fig. 1S). The DES loading was confirmed by 1H-NMR in all cases (Fig. 1; integration o* vs q* and p** vs r**). Block-DES polyacetals with an adequate water solubility were able to be obtained with a drug payload greater than that obtained for the tert-DES (from 2 to 9 wt.%) with a free drug content always b 0.5 wt.% of total drug, a Mw ranging from 26000 g/mol to 35000 g/mol and a moderate Mw/Mn from 1.3 to 1.6 as determined by GPC in THF (Table 1).

drug release rate in both designs, being more important for blockDES family. At acidic pH, block-DES presented a slightly faster drug release rate in comparison to its tert-DES counterpart (1b vs 2b; Fig. 2A vs B). More importantly, differently from tert-DES, block-DES showed what could be considered a ‘biphasic release profile’ typically observed in biodegradable nanoparticulate systems [37,38]. This biphasic pattern in block-DES can more clearly be seen in those polyacetals with greater drug loading (2c > 2b > 2a) (Fig. 2D). As these systems are designed for intravenous (i.v.) administration it is important to explore conjugate stability in plasma trying to mimic physiological conditions. Both tert-DES and block-DES showed non-significant drug release up to 24 h incubation (see supporting information, Fig. 2S).

3.2. pH-dependent degradation of DES-polyacetals 3.3. Biological evaluation in prostate cancer cell models Essential characteristics for polymer–drug conjugates are stability during blood circulation and the capability for drug release from the carrier under selected physiological triggers. Therefore, in order to show the applicability of this approach, it was essential to determine pH-responsiveness of the conjugates under conditions encountered in a biological environment. The pH-dependent degradation profile for DES-polyacetals was hypothesized to be similar to that previously described for APEG [12,13], however it was already demonstrated for tert-DES that the presence of the aromatic groups adjacent to the acetal moiety affects polymer degradation rate at acidic pH, being much faster when DES was present in comparison to the parent APEG systems [16]. HPLC (at 280 nm) was used as quantitative method to determine the amount of DES released from block-DES in comparison to tertDES. As expected, a strong pH dependence on polyacetal degradation was observed for both polymers, with % DES released decreasing pH 5.5 > > pH 6.5 > pH 7.4, which is an ideal profile for a lysosomotropic drug delivery route (Fig. 2A, B) [36]. Importantly, for both polymers the DES was released predominantly as the active isomer of transDES (>80%, data not shown) [16]. Due to the relative hydrophobic/ hydrophilic ratio depending on polyacetal DES loading, it is clear that the different DES content could affect conjugate conformation and consequently also influence drug release kinetics. In fact, as it can be seen in Fig. 2 C, D, a greater DES loading yielded to a slower

Once the polyacetalic systems were synthesized and characterized, their evaluation as anticancer agents was carried out in selected prostate cancer cell lines. In vitro antitumor activity of DES should preferably be assessed using an estrogen responsive cell line, however, DES also inhibits Bcl-2 protein [19,39], the assembly of microtubular proteins [40,41] and even the telomerase activity [42] and therefore, has been shown to be also cytotoxic in non-estrogen responsive cell lines, such as 3T3 fibroblast [43], MOP [44] or B16F10 melanoma cells [16]. Therefore, the biological evaluation of DESpolyacetals 1 and 2 was carried out in two different human prostate cancer cell lines, namely PC3 (Hormone Independent) and LNCaP (Hormone Dependent). For comparison, in cell viability studies, free DES as sodium salt (NaDES) was used. 3.3.1. Cell uptake studies by live-cell fluorescence confocal microscopy Firstly, differences between tert- and block-DES on cell trafficking were explored. In order to carry out these experiments, fluorescently labeled polyacetal derivatives were required. However, the original tert- or block-DES structures do not provide with any extra anchoring positions. Therefore, a functional pendent chain suitable for drug or dye conjugation was incorporated following the already described approach by Tomlinson et al. [12]. We used Fmoc-protected serinol moieties as co-monomers during the

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pH 6

pH 6 pH 7

80

(%) DES released

(%) DES released

B

pH 5.5

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60

40

pH 7

80

60

40

20

20

0

0 0

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480

600

720

0

120

Time (h)

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360

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Time (h)

C

D 100

100

80

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40 1a 20

1b

(%) DES released

(%) DES released

NANOMEDICINE

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60

40 2a 20

2b

1c 0

2c 0

0

72

144

216

288

360

Time (h)

0

72

144

216

288

360

Time (h)

Fig. 2. pH-Dependent DES release from: (A), tert-DES 1b at pH 7.4, 6.5 and 5.5; (B), block-DES 2b at pH 7.4, 6.5 and 5.5; (C), tert-DES 1a-c at pH 5.5, (D) block-DES 2a-c at pH 5.5. The results show the percentage of DES released from total at each time point. Mean values ± SD (n = 3).

polymerization procedure, after deprotection with piperidine aminopendant DES polyacetals were successfully obtained as tert- or blocksystems (basic conditions preserve the acetal bonds already formed as previously demonstrated) (Scheme 3S). 1H-NMR spectra (Fig. 3S) for conjugate tert-DES-Ser 3 and block-DES-Ser 4 confirmed the presence of the serinol within the polymer mainchain with bonds at 4.5 ppm (d, Fmoc-CH-CH2-), 4.7 ppm (t, Fmoc-CH-CH2-) and at 7.3–7.8 ppm from the aromatic entity signals in the Fmoc. The presence of these Fmoc-related signals allows serinol loading quantification; tert-DES-Ser 3 (4.5 wt.% DES, 3.0 wt.% NH2) and block-DES-Ser 4 (4.7 wt.% DES, 3.0 wt.% NH2) (Table 1). After the piperidine deprotection step, an absence of the aromatic peaks from the Fmoc protecting group and the presence of the two distinct sets of acetalic peaks in the 1H-NMR spectra (Fig. 3S) ensured the desired structures and the efficiency of the reaction. By means of an NHS-activated oregon green (OG) dye, fluorescently labeled polyacetals (tert-DES-Ser-OG 5 and block-DES-Ser-OG 6) were then tested by confocal fluorescence live-cell imaging in order to explore differences in cell uptake (Scheme 4S). As has been previously described, live-cell confocal fluorescence microscopy experiments allow better characterization of conjugate cell trafficking by avoiding any fluorescence artifact induced by fixation protocols [45–48]. Low membrane-associated fluorescence was observed in all cases studied at the different incubation times. Both conjugates enter the cell by the endocytic route as demonstrated by the observed colocalization with the lysosomal marker dextran-texas red (Fig. 3). In general, LNCaP cells seems to have slightly higher uptake rate compared with PC3 cells; and even more interestingly, the percentage of

block-DES inside the cells is greater than that observed for tert-DES in both cell lines (Fig. 3). 3.3.2. In vitro cytotoxicity of DES-polyacetals Probably due to the androgen-dependent character, DES derivatives displayed greater cytotoxicity against LNCaP than against PC3 cell line (Fig. 4). Encouragingly, Block-DES IC50 values were nonsignificantly different from those obtained with NaDES (IC50 value for 2b = 0.052 and 0.049 mg/mL, DES-equiv. IC50 value for NaDES = 0.060 and 0.044 mg/mL, DES-equiv. against PC3 and LNCaP, respectively), even following a different cell pharmacokinetics (endocytosis for block-DES vs. diffusion for free DES) (Fig. 4A, B). This could be due to the enhancement of DES water solubility prior to its intracellular release following endocytic uptake. More importantly, block-DES 2 showed greater cytotoxicity than tert-DES 1 in both cell lines (IC50 value for 2b = 0.052 and 0.049 mg/mL, IC50 value for 1b = 0.172 and 0.070 mg/mL, DES-equiv. against PC3 and LNCaP, respectively). The combination of the two key features already described, a slightly faster and biphasic drug release profile at acidic pH from block-DES (Fig. 2) and/or its probably greater cell uptake when compared to tert-DES (Fig. 3) could justify this behavior. The inherent cytotoxicity of the DES-polyacetals not only depend on monomer arrangement, but also on total DES content, being significantly different when loadings below 3 wt.% are compared with those above. This effect seems to be more important in the androgen-independent PC3 cells (Fig. 4C, D) than in the hormonedependent LNCaP cells, indicating the complexity of DES molecular mechanisms of action. Further studies out of the scope of this paper are currently being carried out in this direction.

297

A Tert-DES-Ser-OG

Block-DES-Ser-OG

B Tert-DES-Ser-OG

Block-DES-Ser-OG

Fig. 3. Confocal microscopy images were taken from live: (A) PC3 cells, (B) LNCaP cells after 2 h incubation with tert-DES-Ser-OG 5 and block-DES-Ser-OG 6. Dextran-Texas Red was employed as lysosomal marker (in red) and polymers were labeled with OG (in green, left panel). Co-localization is seen in yellow (central panel).

Finally it is important to note that, as already described for tert-DES [16], block-DES-polyacetals also displayed much lower hemolytic activity than their free counterpart (3%, 4% and 90% Hb released in 1 h; 5%, 4% and 100% Hb released in 24 h for 1,

2 and NaDES, respectively, at 2 mg/ml DES-equiv.) [16] (supporting information Fig. 4S)). This proofs the suitability of these nanoconjugates for i.v. injection in subsequent in vivo studies.

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B

120 110

tert-DES 1b

100

block-DES 2b

Cell viability (%)

*

60

*

50 40

70 60 50 40 30

20

20

10

10 0,10

0 0,01

1,00

Concentration (mg/ml, DES-equiv.)

C

D

110

1b

80

1c

70 60 50 40 30

Cell viability (%)

1a

90

120 100

2a

90

2b

80

2c

70 60 50 40 30

*

20

* *

10

0 0,01

1,00

110

100

10

0,10

Concentration (mg/ml, DES-equiv.)

120

20

NaDes

80

30

0 0,01

block-DES 2b

90

80 70

tert-DES 1b

100

NaDes

*

90

120 110

Cell viability (%)

A

Cell viability (%)

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0,10

1,00

Concentration (mg/ml, DES-equiv.)

0 0,01

*

* 0,10

1,00

Concentration (mg/ml, DES-equiv.)

Fig. 4. Cytotoxicity of DES derivatives measured by MTT assay after 72 h incubation. Tert-DES vs block-DES in (A) PC3 cells and (B) LNCaP cells. DES content influence on cytotoxicity against PC3 cells (C) tert-DES 1a-c, (D) block-DES 2a-c. Data are expressed as mean ± SD (n ≥ 3). *p b 0.05.

3.4. Investigating conformation in solution with DES-polyacetals 1 and 2 In order to better explain the results obtained in the cell models used it was hypothesized that the conformation adopted by the polyacetals in solution could be responsible for the differences observed in drug-release kinetics, cell uptake and therefore cell viability. The size and shape of nanoconjugates in solution are critical aspects for cellular internalization and drug release kinetics from the polymer carrier [31,49,50]. Amphiphilic block copolymers in aqueous solution are known to form aggregates or self-assemble into nanosized particles (e.g., micelles or polymersomes). This is an important feature to take into account for biomedical applications as subtle differences in size and/or shape could significantly alter conjugate therapeutic value. As the main purpose of this paper is the comparison between monomer arrangements, the resulting consequences in the adopted conjugate conformation in solution and ultimately in polyacetal biological output, only polyacetals containing ~4 wt.% DES loading (namely, tert-DES 1b and block-DES 2b) were selected to carry out the experiments described below. 3.4.1. Determination of solution behavior: TEM, DLS, PGSE-NMR and SANS Due to presence of hydrophobic moieties within the structures, systems, characterization of any concentration dependent aggregation behavior was first carried out. The critical aggregation concentrations (CAC), (akin to the critical micelle concentration (CMC) of conventional small-molecule surfactants), were determined using two fluorescent probes; diphenylheptatriene (DPH) and pyrene. In

both cases multiple CACs were found, with that obtained for blockDES CAC much lower than for tert-DES in all cases (i.e. first tert-DES 1b CAC = 0.7 mg/mL; first block-DES 2b CAC = 0.1, mg/mL) (see supporting information Fig. 5S). All subsequent studies were carried out at solution concentrations above the previously determined CAC value, and under conditions designed to mimic physiological environment (salt buffer, pH and temperature). The conformations adopted by the conjugates in solution (size and shape) were studied by transmission electron microscopy (TEM) [51,52], dynamic light scattering (DLS) [51,53] and small-angle neutron scattering (SANS) [30,31]. Using these techniques, marked differences were found between tert-DES and block-DES. TEM allowed the observation of well-defined particles of approximately 100 nm diameter for block-DES (Fig. 5A, Fig. 6S) in high abundance at a solution concentration of 3 mg/ml. This particle size and the stability of the aggregates formed were confirmed by DLS measurements at different conjugate concentrations (1 mg/mL and 3 mg/mL, always above the CAC) (Fig. 5B). In contrast to the block copolymer, it was not possible to obtain an indication of particle size for tert-DES, by either TEM or DLS (Fig. 6S). An indication of the relative particle sizes was obtained by PGSENMR measurements, which have the benefit of being nonperturbative, ensuring any structures formed by the tert-DES conjugate are not disrupted by the measurement. Fig. 6 shows PGSE-NMR data for 10 mg/mL solutions of tert-DES and block-DES, plotted according to Eq. (1) (see materials and methods section), using the normalized signal intensity. Presented in this manner, the difference in slopes indicates a clear difference in the obtained self-diffusion

A

299

B Size: 128 nm Pdi: 0.2

100 nm Fig.5. (A) Transmission electronic micrograph of block-DES at 3 mg/ml. (B) Hydrodynamic diameter of block-DES determined by DLS (size average, n ≥ 3).

model, with the same dimensions as those present in the sample at C > CAC (100 nm length, 2 nm diameter). These characterization data indicate that the molecular structure of the conjugate has a significant effect on the solution behavior; even at the same overall acetal and DES contents, i.e., the segregated arrangement of the DES units can induce sufficient amphiphilic nature in the molecule to induce the formation of large aggregate structures. This is further indicated by a parallel study in methanol rather

A 100

Scattering Intensity, I(Q) / cm-1

rates between the two conjugates, with the tert-DES conjugate (Ds = 2.72 × 10 − 11 m 2 s − 1) moving more slowly than the block-DES counterpart (Ds = 5.87 × 10 − 11 m 2 s − 1) indicating clearly that different solution structures are formed in each case. In order to further elucidate the structure of the aggregates or nanoparticles that are formed, tert- 1b and block-copolymer 2b conjugates containing a fixed DES content of 4 wt.% and were studied by SANS in both PBS (Fig. 7A) and methanol solutions (Fig. 7B) using deuterated solvents to provide contrast. In PBS a significant difference between scattering profiles from the two conjugates is evident, suggesting a difference in particle morphology between the two conjugates in this solvent. For the tert-polymer, the modeling analysis indicates the presence of a single species in solution, which was best fitted by a model for a solid disk (diameter 20 nm, thickness 2.5 nm). This indicates that there is some aggregation of the tert-DES, as indicated by the fluorescence studies. By comparison it was not possible to model the block-copolymer data for the PBS solution using a single species, rather the analysis suggests that two sets of co-existing rods are present in solution, one comparatively long and thin (approximately 100 nm in length, 2 nm in diameter), the other more disk-like (80 nm diameter, 10 nm thick) being the second one more abundant and consistent with the size of the structures observed by TEM and DLS, and significantly larger than the structures indicated by SANS for the tert-DES. A second solution of the block-DES in PBS was studied at a concentration below the CAC (0.008 wt.%), and this was accurately described by a single rod

10

1

0.1

0.01 0.01

0.1

Scattering vector, Q / Å-1

B Scattering intensity, I(Q) / cm-1

Normalised signal

1

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0.01 0.0

1

0.1

0.01 2.0e+6

4.0e+6

6.0e+6

8.0e+6

1.0e+7

1.2e+7

1.4e+7

-2

K / cm s Fig. 6. Normalized PGSE-NMR data from 1 wt.% conjugate solutions at 25 °C; tert-DES in D2O (filled circles) and methanol (open circles); block-DES in D2O (filled squares) and methanol (open squares).

0.001

0.01

0.1

Scattering vector, Q / Å-1 Fig. 7. SANS from 1 wt.% conjugates solutions in (A) D2O. Lines are best-fits to the data as described in the text. (B) MeOD. Solid lines are best-fits to a rod model. Tert-DES (triangles), block-DES (squares), dilute block-DES solution (circles).

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than PBS as shown in Figs. 6 (PGSE-NMR) and 7B (SANS). The SANS data for the two conjugates in methanol were fitted to the same model of a single short rod (1.5 nm diameter, 18.5 nm in length) with an attractive structure factor required to account for the scattering at low Q for block-DES. Both polyacetal and DES are methanol soluble, hence the solvent has a similar affinity for both components of the conjugate. From the similarity between data obtained for the two conjugates by both SANS and PGSE-NMR data (Figs. 6 and 7), it is evident that the loss of the solvophobicity of the block-DES on switching from PBS to methanol is sufficient to remove the driving force for aggregation of the DES-rich region of the molecule observed in D2O/PBS, and with it the large difference in solution structures observed between the block-DES and tert-DES conjugates. Hence the solution behavior in aqueous media is driven by the hydrophobicity of the DES. It was thus determined that, compared to the tert-DES polymer, block-DES presents a more stable and better defined particulate conformation in the nanosized range, that could explain its different biological behavior in prostate cancer cell models. 4. Conclusions Here we describe a new strategy for the synthesis of rationally designed pH-responsive conjugates in order to improve the properties of the first generation DES-polyacetals. These block-co-polymer derivatives showed more controlled architecture than the tert-polymer, with higher drug capacity maintaining aqueous solubility. The conformation of both polymers adopted in solution was different due to the amphiphilic character of the block-co-polymer, indicated by a different CAC in both systems. Both, tert- and block-DES, showed clear pH-dependent drug release kinetics, however drug release profile was significantly different with a slightly greater DES release and a ‘biphasic’ mode for block-DES, indicative of the presence of particulate assembles. After exhaustive conformational studies by several techniques including SANS and PGSE-NMR, it was demonstrated that the molecular structure of the conjugate has a significant effect on the solution behavior; even at the same overall acetal and DES contents. In aqueous solution tert-DES is present as single system with an approximate diameter of 20 nm with 2 nm thickness. On the other hand, for block-DES two sets of coexisting species are present, one with similar shape but greater length than tert-DES (100 nm length, 2 nm thickness) and a second and more abundant system with a disk-like conformation (80 nm diameter, 10 nm thickness). The different release kinetics together with a greater cell uptake probably induced by a more spherical shape [5,49,54,55], yielded to an enhancement in cytotoxicity for block-DES in selected prostate cancer cell lines. Not only monomer arrangement but also DES loading showed a significant effect on polyacetal cytotoxicity. Our results and others already reported [4,29,49,54,56] ratify the impact of conjugate solution properties on biological behavior and therefore nanomedicine therapeutic output. Acknowledgments The authors thank the Spanish Ministry of Science and Innovation (MICINN) (RYC-2006-002438, CTQ2007-60601, CTQ2010-18195/ BQU), Cardiff University and Centro de Investigación Príncipe Felipe (Valencia, Spain) for financial support. The authors also thank the EPSRC Platform (EP/CO13220/1) for travel support and both STFC and Institute Laue-Langevin for beam time allocation and consumables funding. Richard Heenan (ISIS) is thanked for advice regarding the data modeling. I. Abasolo, Y. Fernández and S. Schwartz Jr are thanked for helpful discussions on conjugate biological applications. C. Scholz, M.R. Onuoha and J. Pendas are thanked for technical support.

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