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A modular synthetic approach for adjusting the disassembly rates of enzyme-responsive polymeric micelles Assaf J Harnoy, Marina Buzhor, Einat Tirosh, Rona Shaharabani, Roy Beck, and Roey J. Amir Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01906 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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A Modular Synthetic Approach For Adjusting The Disassembly Rates Of Enzyme-Responsive Polymeric Micelles Assaf J. Harnoy,a,b Marina Buzhor,a,b Einat Tirosh,b,c Rona Shaharabani,b,c Roy Beckb,d and Roey J. Amira,b,e,*

a

Department of Organic Chemistry, School of Chemistry, Faculty of Exact Sciences, Tel-Aviv

University, Tel-Aviv 6997801, Israel b

Tel-Aviv University Center for Nanoscience and Nanotechnology, Tel-Aviv University, Tel-

Aviv 6997801, Israel c

Department of Physical Chemistry, School of Chemistry, Faculty of Exact Sciences, Tel-Aviv

University, Tel-Aviv 6997801, Israel d

School of Physics and Astronomy, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv

6997801, Israel e

Blavatnik Center for Drug Discovery, Tel-Aviv University, Tel-Aviv 6997801, Israel

ACS Paragon Plus Environment

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KEYWORDS Stimuli-responsive block copolymers, enzyme-responsive polymers, polymeric micelles, biodegradable polymers, dendrimers.

ABSTRACT: Self-assembled nano-structures and their stimuli-responsive degradation have been recently explored to meet the increasing need for advanced biocompatible and biodegradable materials for various biomedical applications. Incorporation of enzymes as triggers that can stimulate the degradation and disassembly of polymeric assemblies may be highly advantageous owing to their high selectivity and natural abundance in all living organisms. One of the key factors to consider when designing enzyme-responsive polymers is the ability to fine-tune the sensitivity of the platform towards its target enzyme in order to control the disassembly rate. In this work, a series of enzyme-responsive amphiphilic PEG-dendron hybrids with increasing number of hydrophobic cleavable end-groups was synthesized, characterized and compared. These hybrids were shown to self-assemble in aqueous media into nano-sized polymeric micelles, which could encapsulate small hydrophobic guests in their cores and release them upon enzymatic stimulus. Utilization of dendritic scaffolds as the responsive blocks granted ultimate control over the number of enzymatically cleavable end-groups. Remarkably, as we increased the number of end-groups, the micellar stability increased significantly and the range of enzymatic sensitivity spanned from highly responsive micelles to practically non-degradable ones. The reported results highlight the remarkable role of hydrophobicity in determining the micellar stability towards enzymatic degradation and its great sensitivity to small structural changes of the hydrophobic block, which govern the accessibility of the cleavable hydrophobic groups to the activating enzyme.


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Introduction Stimuli-responsive amphiphilic block copolymers have recently attracted considerable attention due to their ability to self-assemble in aqueous media into smart polymeric micelles.1–4 The inner hydrophobic cores of such micelles can be exploited to encapsulate small, poorly soluble guest molecules and release them on cue.5,6 These unique features grant polymeric micelles the potential to be crafted for various biomedical applications such as controlled drug delivery,7 bio-sensing and diagnostics,8 tissue engineering,9 etc. To this end, there have been many reports on stimuli-responsive polymeric micelles that can respond to various types of stimuli such as pH,10 temperature,11,12 irradiated light,13,14 redox conditions15,16 or their combinations.17–21 However, polymeric micelles that can respond to variations in enzymatic activity are not as prominent in the literature.8,22–24 Utilization of enzymes as potential triggers for disassembly of polymeric micelles can be highly advantageous due to the catalytic efficiency and high selectivity of the activating enzymes towards their substrates.25 Moreover, overexpression of specific enzymes is frequently associated with specific diseases and may be picked up locally by cleverly designed micelles to induce a site-specific release of their molecular payload.26 We recently reported an accelerated and simple synthetic approach for preparation of highly modular amphiphilic polymer-dendron hybrids.27–31 This unique molecular architecture, which was introduced by Frechet, Gitsov, Wooley and Hawker in the early 1990’s, enables extremely high structural precision due to the monodisperse nature of the dendritic block.32–34 The molecular design of our hybrids included a mono-functional hydrophilic polyethylene glycol (PEG) chain conjugated to a symmetrical dendron unit with four hydrophobic enzymeresponsive end-groups.27 These amphiphilic hybrids self-assembled in aqueous media into spherical polymeric micelles that disassembled in response to the enzymatic hydrolysis of the


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hydrophobic end-groups. Comparing hybrids with three different lengths of PEG chains and the same dendron unit, allowed us to demonstrate that the length of the PEG chain may be adjusted to fine-tune the disassembly rate of the formed micelles in response to the enzymatic activation.27 Furthermore, the correlation between the increase in the critical micelle concentration (CMC) and faster enzymatic degradation rates as the PEG blocks got longer, led us to conclude that the enzymatic hydrolysis of the hybrids occurs at the monomeric state, which is in equilibrium with the assembled micellar state.

Following these findings, we wished to

investigate how changes in the hydrophobicity of the dendron block would affect the supramolecular properties of the formed micelles and their sensitivity towards the enzymatic stimulus. We postulated that increasing the number of hydrophobic end-groups, while keeping the PEG chain constant, would increase the spontaneous tendency of the hybrids to selfassemble. As a result, the micellar stability was expected to increase gradually, while the disassembly rate upon enzymatic activation was expected to decrease (Figure 1).


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Figure 1. Micellar stability towards enzymatic activation can be adjusted by tuning the number of dendrons and/or end-groups. Experimental section Instrumentation. High Pressure Liquid Chromatography (HPLC): All measurements were recorded on a Waters Alliance e2695 separations module equipped with a Waters 2998 photodiode array detector. All solvents and Trifluoroacetic acid (TFA) were purchased from BioLab Chemicals and were used as received. All solvents are HPLC grade. 1H and

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C NMR:

spectra were recorded on Bruker Avance I and Avance III 400 MHz spectrometers as indicated. Chemical shifts are reported in ppm and referenced to the solvent. The molecular weights of the PEG-dendron hybrids were determined by comparison of the areas of the peaks corresponding to the PEG block (3.63 ppm in CDCl3) and the protons peaks of the dendrons. GPC: All measurements were recorded on Viscotek GPCmax by Malvern using refractive index detector and PEG standards (purchased from Sigma-Aldrich) were used for calibration. Infrared spectra: All measurements were recorded on a Bruker Tensor 27 equipped with a platinum ATR diamond. Fluorescence spectra: All measurements were recorded on an Agilent Technologies Cary Eclipse Fluorescence Spectrometer using quartz cuvettes. CMC: All measurements were recorded on a TECAN Infinite M200Pro device. Matrix Assisted Laser Desorption Ionization (MALDI-TOF) MS: Analysis was conducted on a Bruker AutoFlex MALDI-TOF MS (Germany) and also on a Waters MALDI synapt (USA). DHB matrix was used. Transmission Electron Microscopy (TEM): Images were taken by a Philips Tecnai F20 TEM at 200 kV, Dynamic Light Scattering (DLS): All measurements were recorded on a Corduan Technology VASCOγ particle size analyzer. Materials. Poly (Ethylene Glycol) methyl ether (Mn = 5 kDa), 2-(Boc-amino)-ethanethiol, 2,2-


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dimethoxy-2-phenylacetophenone (DMPA), Penicillin G Amidase from Escherichia coli (PGA), Allyl bromide, Propargyl bromide (80% in toluene), N,N'-dicyclohexylcarbodiimide (DCC), 4dimethylaminopyridine (DMAP), phenylacetic acid and Sephadex® LH20 were purchased from Sigma-Aldrich. Cystamine hydrochloride, potassium hydroxide and Diisopropylethylamine (DIPEA) were purchased from Merck. Gallic acid was purchased from Chem Impex. 3,5 dihydroxy benzoic acid was purchased from Apollo Scientific Ltd. Anhydrous potassium carbonate was purchased from J. T. Baker. Silica Gel 60Å, 0.040-0.063mm, Sodium Hydroxide and all solvents were purchased from Bio-Lab and were used as received. Deuterated solvents for NMR were purchased from Cambridge Isotope Laboratories (CIL), Inc. Synthesis of hybrids. mPEG5kDa-NH2. mPEG5kDa-allyl and mPEG5kDa-NH2 were prepared as was described in previously reported procedure. 29 mPEG5kDa-propargyl. Methoxy poly(ethylene glycol) (Mn = 5 kDa, 5.00 g, 1 mmol), KOH (1.68 g, 30 mmol, 30 eq.) and 80% propargyl bromide in toluene (3.25 mL, 30 mmol, 30 eq.) were reacted in toluene (50 mL) following a previously reported procedure.27,29 The product was obtained as off-white to light yellowish solid in quantitative yield (4.97 g). 1H-NMR (CDCl3): δ 4.17 (d, J = 2.4Hz, 2H, -O-CH2-C≡), 3.78-3.46 (m, 505H, PEG backbone), 3.35 (s, 3H, H3C-O-), 2.43 (t, J = 2.4Hz, 1H, -C≡CH); 13C-NMR (CDCl3): δ 74.6, 72.0, 70.6, 69.2, 59.1, 58.5; FT-IR, ν(cm-1): 2883, 1558, 1541, 1522, 1508, 1472, 1466, 1457, 1360, 1319, 1280, 1241, 1146, 1106, 1061, 962, 842; GPC (DMF + 25 mM NH4Ac): Mn = 5.0 kDa, PDI = 1.03, Expected Mn = 5.0 kDa. mPEG5kDa-(NH2)2. mPEG5kDa-propargyl (1.26 g, 0.25 mmol), cysteamine hydrochloride (1.14 g, 10 mmol, 40 eq.) and DMPA (26 mg, 0.1 mmol, 0.4 eq.) were reacted in MeOH (6 mL) following a previously reported procedure.29 The product was obtained as an off-white solid in


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82% yield (1.06 g). 1H-NMR (CDCl3): δ 3.79-3.41 (m, 503H, PEG H), 3.34 (s, 3H, H3C-O-), 2.96-2.61 (m, 11H, -S-CH2- + - CH2-NH2);

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C-NMR (CDCl3): δ 73.3, 72.0, 70.6, 59.1, 45.8,

41.7, 41.3, 37.4, 35.9, 34.8; FT-IR, ν(cm-1): 2883, 1558, 1541, 1521, 1508, 1472, 1457, 1360, 1342, 1280, 1241, 1147, 1102, 1061, 962, 843; GPC (DMF + 25 mM NH4Ac): Mn = 5.3 kDa, PDI = 1.03, Expected Mn = 5.3 kDa. General procedure for conjugation of mPEG5kDa-NH2 / mPEG5kDa-(NH2)2 to PNP-ABx. mPEG5kDa-NH2 or mPEG5kDa-(NH2)2 were dissolved in DCM:DMF 1:1v/v (1 mL per 100 mg PEG). DIPEA (10 eq. per amine) and the desired 4-nitrophenyl ester of the branching unit (PNPABx, 3 eq. per amine) were added and the reaction was allowed to stir overnight at room temperature. Then, the reaction mixture was loaded as-is on a LH20 (Sephadex®) size exclusion column and eluted with MeOH. Fractions that contained the product (identified by UV light and/or staining with Iodine) were unified and MeOH was evaporated to dryness. In order to facilitate the solidification of the product, the oily residue was redissolved with DCM (1 mL per 100 mg PEG) and hexane (5 mL per 100 mg PEG) was added. Organic solvents were evaporated to dryness and the obtained solid was dried under high vacuum. General procedure for thiol-ene/yne reaction with 2-(Boc-amino)ethanethiol. The PEG reactant, 2-(Boc-amino)ethanethiol (20 eq. per double bond or 40 eq. per triple bond) and DMPA (1 mol % with respect to the thiol) were dissolved in MeOH (0.5 mL per 100 mg PEG). The solution was purged with N2 for 15 minutes and then placed under UV light (365 nm) for 2 hours. The product was isolated and purified as described above in the conjugation procedure. General procedure for Boc removal and amidation with 4-nitrophenyl 2-phenylacetate. The PEG reactant was dissolved in DCM (0.5 mL per 100 mg PEG) and TFA was added (0.5 mL per 100 mg PEG). After 30 minutes, all organic solvents were evaporated to dryness and the


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obtained oily residue was dried under high vacuum for 1 hour. Then, the deprotected PEG was redissolved with DCM:DMF 1:1v/v (1 mL per 100 mg PEG), DIPEA (10 eq. per amine) and 4nitrophenyl 2-phenylacetate27 (3 eq. per amine) were added, and the reaction was allowed to stir overnight at room temperature. The product was isolated and purified as described above in the conjugation procedure. The synthesis of all intermediate hybrids appears in the SI and hybrid 2 was synthesized as previously described.27 mPEG5kDa-dend-(3Ph) (Hybrid 1). The Boc groups of mPEG5kDa-dend-(3Boc) (118 mg, 0.02 mmol) were removed with TFA and then the deprotected PEG was reacted with DIPEA (95 µL, 0.54 mmol, 30 eq.) and 4-nitrophenyl 2-phenylacetate (46 mg, 0.18 mmol, 9 eq.) in DCM:DMF 1:1v/v (1.2 mL) according to the described general procedure. The product was obtained as a white solid in 90% yield (107 mg). 1H-NMR (CDCl3): δ 7.17-7.25 + 7.27-7.36 (m, 15H, arom H), 7.11 (t, J = 5.5Hz, 1H, -NHCO-Ar), 7.04 (s, 2H, arom H), 6.17 (t, J = 5.6Hz, 1H, -NH-CO-), 6.08 (t, J = 5.3Hz, 2H, -NHCO-), 4.06 (t, J = 6.1Hz, 4H, Ar-O-CH2-), 4.01 (t, J = 5.8Hz, 2H, ArO-CH2-), 3.55-3.81 (m,488H, PEG backbone), 3.33-3.40 (m, 9H, H3C-O + -CH2-NH-CO-), 2.522.77 (m, 16H, -CH2-S-), 2.00 (qui, J = 6.5Hz, 4H, -O-CH2-CH2-CH2-S-), 1.81-1.93 (m, 4H, -OCH2-CH2-CH2-S-); 13C-NMR (CDCl3): δ 171.3, 171.2, 167.1, 152.6, 140.6, 135.0, 134.9, 130.1, 129.5, 129.02, 128.97, 127.4, 127.3, 106.3, 72.0, 71.7, 70.7, 70.3, 69.6, 67.5, 59.1, 43.79, 43.76, 39.4, 39.0, 38.9, 31.8, 31.7, 31.6, 30.4, 29.8, 29.5, 28.4, 28.3; FT-IR, ν(cm-1): 2868, 1569, 1558, 1541, 1522, 1508, 1497, 1489, 1473, 1457, 1437, 1419, 1396, 1374, 1348, 1277, 1248, 1146, 1093, 1039, 949, 845; GPC (DMF + 25 mM NH4Ac): Mn = 5.9 kDa, PDI = 1.04, Expected Mn = 6.0 kDa.


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mPEG5kDa-dend-(6Ph) (Hybrid 3). mPEG5kDa-dend-(6Boc) (148 mg, 0.023 mmol) was deprotected with TFA and then reacted with DIPEA (220 µL, 1.24 mmol, 54 eq.) and 4nitrophenyl 2-phenylacetate (106 mg, 0.41 mmol, 18 eq.) as described in the general procedure. The product was obtained as a white solid in quantitative yield (145 mg). 1H-NMR (CDCl3): δ 7.71 (t, J = 5.1Hz, 1H, -NH-CO-Ar), 7.11-7.25 + 7.27-7.36 (m, 32H, arom H), 6.80 (t, J = 5.0Hz, 1H, -NH-CO-), 6.69 (t, J = 5.2Hz, 1H, -NH-CO-), 6.59 (t, J = 5.2Hz, 2H, -NH-CO-), 6.51 (t, J = 5.4Hz, 2H, -NH-CO-), 3.99-4.31 (m, 6H, Ar-O-CH2-), 3.50-3.81 (m, 523H, PEG backbone), 3.24-3.40 (m, 15H, H3C-O- + -CH2-NH-CO-), 2.48- 3.18 (m, 25H, -CH2-S- + -CH-S-), 1.83 (qui, J = 6.7Hz, 2H, -O-CH2-CH2-CH2-S-);

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C-NMR (CDCl3): δ 171.7, 171.6, 166.9, 151.9, 140.0,

135.2, 135.1, 130.4, 129.4, 128.9, 128.82, 128.80, 127.25, 127.22, 127.15, 127.13, 72.0, 70.6, 70.2, 69.7, 59.1, 46.1, 45.0, 43.63, 43.59, 43.55, 39.8, 39.7, 39.5, 39.3, 34.5, 32.5, 32.3, 31.5, 31.0, 29.7, 28.5; FT-IR, ν(cm-1): 2866, 1569, 1558, 1540, 1522, 1507, 1496, 1488, 1472, 1457, 1437, 1419, 1397, 1366, 1348, 1277, 1246, 1146, 1102, 1040, 949, 845; GPC (DMF + 25 mM NH4Ac): Mn = 6.5 kDa, PDI = 1.06, Expected Mn = 6.6 kDa. mPEG5kDa-[dend-(3Ph)]2 (Hybrid 4). mPEG5kDa-[dend-(3Boc)]2 (187 mg, 0.028 mmol) was deprotected with TFA and then reacted with DIPEA (285 µL, 1.65 mmol, 60 eq.) and 4nitrophenyl 2-phenylacetate (127 mg, 0.5 mmol, 18 eq.) as described in the general procedure. The product was obtained as an off-white solid in quantitative yield (188 mg). 1H-NMR (CDCl3): δ 8.15-8.36 (m, 2H, -NH-CO-), 7.27-7.35 + 7.17-7.25 (m, 30H, arom H), 7.11 (s, 4H, arom H), 6.50-6.59 (m, 4H, -NH-CO-), 6.27-6.40 (m, 2H, -NH-CO-), 3.96 (t, J = 5.7 Hz, 4H, para Ar-O-CH2-), 3.83-3.92 (m, 8H, meta Ar-O-CH2-), 3.54-3.76 (m, 482H, PEG backbone), 3.29-3.39 (m, 15H, H3C-O- + -CH2-NH-CO-), 3.02-3.19 (m, 1H, -CH-S-), 2.49- 2.94 (m, 30H, CH2-S-), 1.78-1.98 (m, 12H, -O-CH2-CH2-CH2-S-);

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C-NMR (CDCl3): δ 171.5, 171.3, 167.3,


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167.2, 152.6, 140.4, 135.0, 129.7, 129.5, 129.0, 127.4, 127.3, 106.2, 73.0, 72.0, 71.7, 67.4, 59.1, 43.7, 39.0, 31.7, 31.6, 30.4, 29.4, 28.4, 28.3; FT-IR, ν(cm-1): 2866, 1570, 1559, 1540, 1520, 1508, 1496, 1487, 1473, 1457, 1432, 1419, 1398, 1367, 1350, 1277, 1246, 1146, 1095, 1040, 944, 845; GPC (DMF + 25 mM NH4Ac): Mn = 6.9 kDa, PDI = 1.05, Expected Mn = 6.9 kDa. mPEG5kDa-[dend-(4Ph)]2 (Hybrid 5). mPEG5kDa-[dend-(4Boc)]2 (175 mg, 0.025 mmol) was deprotected with TFA and then reacted with DIPEA (315 µL, 1.8 mmol, 72 eq.) and PNP-PhAc Acid (154 mg, 0.6 mmol, 24 eq.) as described for mPEG5kDa-3Ph. The product was obtained as a white solid in quantitative yield (177 mg). 1H-NMR (CDCl3): δ 7.86-8.16 (m, 2H, -NH-CO-), 7.11-7.43 (m, 40H, arom H + CHCl3), 6.99 (s, 4H, arom H), 6.21-6.74 (m, 10H, arom H + -NHCO-), 3.89-4.15 (m, 8H, Ar-O-CH2-), 3.51-3.73 (m, 552H, PEG backbone), 3.44-3.50 (m, 16H, CO-CH2-Ph), 3.20-3.41 (m, 19H, -CH2-NH-CO- + H3C-O-), 2.40-3.15 (m, 35H, -CH2-S- + -CHS-);

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C-NMR (CDCl3): δ 171.5, 171.4, 167.2, 159.4, 136.6, 135.1, 129.4, 128.9, 127.2, 106.4,

105.0, 72.8, 72.0, 71.8, 70.6, 69.7, 59.1, 44.8, 43.6, 39.6, 39.5, 39.2, 39.1, 34.2, 32.2, 31.2; FTIR, ν(cm-1): 2868, 1569, 1560, 1542, 1521, 1509, 1496, 1485, 1473, 1458, 1435, 1420, 1398, 1365, 1348, 1277, 1246, 1145, 1098, 1043, 942, 845; GPC (DMF + 25mM NH4Ac): Mn=7.1kDa, PDI=1.09, Expected Mn = 7.2kDa. mPEG5kDa-[dend-(6Ph)]2 (Hybrid 6). mPEG5kDa-[dend-(6Boc)]2 (120 mg, 0.015 mmol) was deprotected with TFA and then reacted with DIPEA (315 µL, 1.8 mmol, 120 eq.) and 4nitrophenyl 2-phenylacetate (142 mg, 0.55 mmol, 36 eq.) as described in the general procedure. The product was obtained as an off-white solid in 93% yield (115 mg). 1H-NMR (CDCl3): δ 8.03-8.41 (m, 2H, -NH-CO-), 7.03-7.50 (m, 64H, arom H), 6.48-6.99 (m, 12H, -NH-CO-), 3.884.26 (m, 12H, -Ar-O-CH2-), 3.50-3.88 (m, 534H, PEG backbone), 3.18-3.39 (m, 27H, -CH2-NHCO- + H3C-O-), 2.39-3.15 (m, 49H, -CH2-S- + -CH-S-);

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CNMR (CDCl3): δ 171.8, 171.6,


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166.9, 152.0, 139.8, 135.23, 135.19, 135.16, 129.8, 129.4, 128.8, 127.14, 127.10, 106.4, 74.5, 74.0, 72.9, 72.0, 70.6, 69.4, 69.1, 67.1, 63.7, 59.1, 46.1, 45.0, 43.5, 43.4, 39.7, 39.5, 39.3, 34.4, 32.3, 32.2, 30.9; FT-IR, ν(cm-1): 2868, 1570, 1559, 1543, 1522, 1509, 1496, 1485, 1473, 1459, 1435, 1421, 1399, 1365, 1348, 1277, 1248, 1148, 1101, 1043, 944, 844; GPC (DMF + 25 mM NH4Ac): Mn = 8.1 kDa, PDI = 1.12, Expected Mn = 8.1 kDa.

Molecular design To test the effect of the number of dendritic end-groups on the micellar stability towards enzymatic degradation, a series of six amphiphilic PEG-dendron hybrids with increasing number of end-groups (3, 4, 6, 8 and 12) was synthesized, and their self-assembly and enzymatic degradation were studied and compared. Instead of using the “classical” approach of increasing the number of end-groups by synthesizing dendrons of higher generations, we decided to increase the multiplicity of the branching unit. This strategy, which was previously reported by Roy,35,36 Thayumanavan37 and others, allowed us a rapid and systematic growth of the number of dendritic end-groups. Furthermore, it granted the minimization of the degree of structural changes between the different hybrids and allowed us to study dendrons of similar generation but with increasing number of end-groups. As illustrated in Figure 2, all hybrids were composed of a 5kDa PEG chain as their hydrophilic block and differed in the number of dendron units and/or end-groups. Hybrids 1-3 were composed of a single dendron unit with 3, 4 and 6 hydrophobic substrates, respectively. The number of end-groups was doubled for hybrids 4-6 by “growing” two dendron units at the block copolymer junction. The applied synthetic methodology allowed us to minimize the number of synthetic steps and to synthesize all hybrids, regardless of the


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number of end-groups, in overall high yielding three synthetic steps starting from amino- or diamino-methoxy PEG.

Figure 2. Schematic representation of PEG-dendron hybrids 1-6. Blue: Hydrophilic 5kDa PEG chain, Red: Hydrophobic enzyme-cleavable end-groups.

Results and discussion Synthesis of the amphiphilic hybrids 5kDa methoxy PEG (mPEG5kDa-OH) was turned into amino methoxy PEG (mPEG5kDa-NH2) and diamino methoxy PEG (mPEG5kDa-(NH2)2) using two high yielding synthetic steps: First, the hydroxyl chain-end was reacted with allyl/propargyl bromide to yield a double/triple bond, respectively. Second, the double/triple bond was reacted with cysteamine•HCl in a thiol-ene/yne reaction,38–40 respectively, followed by an alkaline treatment with NaOH, to yield the desired mono or diamino PEGs (Scheme 1).


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Scheme 1. Synthesis of amino methoxy PEG and diamino methoxy PEG. In order to obtain dendron units that would eventually lead to a different number of endgroups, three kinds of ABx branching units (x = 3, 4 or 6) were prepared from 3,5 dihydroxy benzoic acid and gallic acid (Scheme 2). The carboxylic acid moiety was later activated as a 4nitrophenyl ester to allow facile and simple conjugation to the polymeric chain-ends.

Scheme 2. ABx branching units (x = 3, 4, or 6) designed for thiol-ene/yne click chemistry. The general synthetic approach for the preparation of the final hybrids included 3 synthetic steps: First, mPEG5kDa-NH2 or mPEG5kDa-(NH2)2 were conjugated to an active 4-nitrophenyl ester of the desired branching unit. Second, the double/triple bonds were reacted with 2-(Bocamino)-ethanethiol through thiol-ene/yne click chemistry, respectively, to afford an accelerated growth to 3, 4, 6, 8 or 12 Boc protected amino groups. Last, the Boc protecting groups were


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removed with TFA and the free amines were further reacted with an active 4-nitrophenyl ester of phenylacetic acid. The synthetic buildup of hybrid 4 is presented in Scheme 3 as an example and the synthetic schemes for the other hybrids can be found in the Supporting Information (Figures S14, S18, S19, S27, S31). PEG-dendron hybrids 1-6 were all obtained as off-white to lightyellow solids with excellent overall yields. The synthesized polymers, branching units and final hybrids were all characterized by 1H- and

13

C-NMR, Gel Permeation Chromatography (GPC),

Infra-Red (IR) and Mass Spectroscopy (MS) in order to confirm their structures, and the experimental values were in good agreement with the theoretical ones (Supporting Information). The GPC overlay of the final hybrids, 1-6, together with the starting PEG (mPEG5kDa-OH), demonstrated the increase in Mn values that were obtained for each hybrid as the dendritic block became larger (Figure 3 and Table 1). In addition, the polydispersity of the final hybrids remained low throughout the synthesis, demonstrating the high molecular precision and structural control of the applied synthetic approach. The molecular weights of the final hybrids were also characterized using MALDI-TOF analysis and correlated well with the theoretical values (Table 1 and Figures S41-S46).


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Scheme 3. Preparation of hybrid 4 from diamino methoxy PEG (mPEG5kDa-(NH2)2).

Figure 3. GPC overlay of hybrids 1-6 and mPEG5kDa-OH starting material. Self-assembly


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In order to evaluate the ability of the amphiphilic hybrids 1-6 to self-assemble into micelles, the PEG-dendron hybrids were dissolved directly in aqueous buffer (phosphate, pH 7.4). Selfassembly was first examined by utilizing the solvatochromic dye Nile Red.41 At low hybrids’ concentrations, no micellar aggregates were formed and as a result, the emission intensity of Nile Red was kept relatively low. However, at higher hybrids’ concentrations, the amphiphilic monomers began to self-assemble and encapsulate the hydrophobic dye within the formed hydrophobic cores. As a result, the emission intensity of Nile Red increased sharply and the CMC was determined as the cross-section of these opposite trends (Figure 4).

Figure 4. CMC determination for hybrids 1-6 using Nile Red fluorescence. These measurements indicated the formation of micellar aggregates with CMC values in the range of 3-50 µM, depending on the hydrophobicity of the dendritic block (Table 1). The following trend was observed: increasing the number of hydrophobic end-groups caused the CMC to decrease, and further addition of yet another dendron unit decreased the CMC even further.


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Next, we measured the sizes of the self-assembled structures using DLS (Figure 5), which indicated the formation of particles with diameters ranging from 20 nm for the smaller hybrids to 30 nm for the larger ones.

Figure 5. DLS measurements of hybrids 1-6 in phosphate buffer (pH 7.4). [Hybrid] = 160 µM. The spherical shape and nanometric size of the micelles were further confirmed using TEM (Figure 6).

Figure 6. TEM images of hybrids 1-6. Scale bar represents 50 nm.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

No. Hybrid Dendrons

No.

Theoretical

Endgroups

Mn [kDa]

Weight ratioa

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GPC

MALDI

Mn

Mn

CMCb Dhc [µM]

PEG: Dendron

[nm]

[kDa] [kDa] (PDI) 5.9

1

3

6.0

83 : 17

6.0

46 ± 3 19 ± 1

6.2

18 ± 1 19 ± 2

6.6

9±2

23 ± 2

7.0

5±1

21 2d

7.2

6±1

27 ± 2

8.0

3±1

29 ± 2

(1.03) 6.0 2

1

4

6.1

82 : 18 (1.03) 6.5

3

6

6.6

76 : 24 (1.03) 6.8

4

6

6.9

72 : 28 (1.03)

±

7.1 5

2

8

7.2

69 : 31 (1.03) 8.0

6

12

8.1

62 : 38 (1.03)

Table 1. Summary of characterizations for hybrids 1-6: a) Weight ratios based on theoretical values; b) CMC was determined using Nile Red fluorescence; c) Micelles' diameter was measured by DLS; d) Larger aggregates were also observed in the DLS data (Figure 5). Comparing the 1H-NMR spectra of the hybrids in CDCl3 and in D2O further supported the formation of self-assembled micelles with core-shell morphology (Figure 7). In CDCl3, a good solvent for both types of blocks, the amphiphilic hybrids did not self-assemble and all of the hybrids' protons were equally visible. However, upon dissolving the hybrids in D2O, most of the monomers self-assembled into micelles and the protons of the dendritic blocks collapsed together to form the hydrophobic cores. As a consequence, the relative mobility of the cores decreased


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and the signals of the dendritic protons were broadened substantially compared to the spectra in CDCl3. This effect resulted in an almost complete disappearance of the dendritic protons and only protons that belonged to the PEG backbone were clearly noticeable. Interestingly, in D2O, the protons that belonged to the phenyl end-groups at 7.0 ppm were slightly visible for hybrid 1 and their signal decreased as we increased the number of phenyl end-groups for hybrids 2 and 3, until they became completely invisible for hybrids 4-6.

Figure 7. a) 1H-NMR spectra overlay of hybrids 1-6 in CDCl3. The signal intensity of phenyl protons increases proportionally to the number of end-groups. b) 1H-NMR spectra overlay of hybrids 1-6 and mPEG5kDa-OH in D2O. The signal intensity of the phenyl end-groups decreases, as their number increases.


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The 1H-NMR spectra supplied us with a first qualitative indication of the relative tendency of hybrids to form micelles. Based on the differences between hybrids 3 and 4, which have the same number of end-groups, it was clear that applying two dendron units per hybrid instead of just one increased the tendency of the hybrids to self-assemble. Furthermore, the relatively lower CMC value that was calculated for hybrid 4 and its tendency to form larger aggregates in addition to micelles, indicated that its specific molecular structure led to a drastic increase of its hydrophobicity and tendency to aggregate. While this trend can be attributed to the addition of another aromatic branching unit, which increases the hydrophobicity of the dendron block, the complete rationalization of its extreme behavior is currently under further studies by exploring molecular dynamics to investigate the molecular origins of the uniqueness of hybrid 4. Enzymatically triggered disassembly After the self-assembly of hybrids 1-6 into micelles was studied and confirmed, we tested their response to enzymatic activation using HPLC, fluorescence of Nile Red and DLS. Using HPLC for time-dependent monitoring enabled us to follow directly the degradation rate of the hybrids and the formation of partially degraded hybrids in response to the activating enzyme, Penicillin G Amidase (PGA),42 which can hydrolyze the phenyl acetamide end-groups. Upon addition of the enzyme, the area of the peak of the starting hybrid began to decrease and partially-cleaved intermediates were observed at shorter retention times. To evaluate the stability of the hybrids in the absence of the enzyme, all hybrids were also incubated in buffer solution and were found to be completely stable under these conditions (Figures S61-S68) A typical HPLC overlay of the degradation of hybrid 3 in the presence of the activating enzyme is presented in Figure 8. The chromatogram of the fully cleaved hybrid, which was synthetically available to our disposal, was placed last as a control.


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Figure 8. HPLC overlay for the enzymatic degradation of hybrid 3. [Hybrid] = 160 µM, [PGA] = 0.1 µM, T = 37 °C, λ = 260 nm. Hybrids 1, 2 and 4 were also tested under similar conditions and their HPLC degradation overlays can be found in the supporting information (Figures S61, S62 and S65). We then plotted the peak areas of hybrids 1-4 as a function of time and observed that their degradation rates slowed down dramatically as the number of end-groups increased from 3 to 6 (Figure 9). In addition, spreading 6 end-groups over two dendron units (hybrid 4) caused the enzymatic degradation to slow down even further.

Figure 9. Enzymatic degradation of hybrids 1-4 by HPLC. [Hybrid] = 160 µM, [PGA] = 0.1 µM, T = 37 °C.


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Since HPLC could not provide any indication on the presence or absence of micelles in the studied solutions, hybrids 1-4 were retested under the same conditions in the presence of Nile Red. Similarly to the CMC determination technique, the emission intensity of Nile Red is sensitive to the polarity of its environment and therefore could be used to monitor the presence of micelles and their disassembly in response to the enzymatic trigger. Hence, fluorescence emission scans were performed periodically after addition of the enzyme (Figures S53-S55 and S57) and the maximum intensity at about 630 nm was plotted as a function of time (Figure 10).

Figure 10. Time-dependent monitoring of Nile Red’s emission intensity in response to the enzymatic activation of the micelles. [Hybrid] = 160 µM, [PGA] = 0.1 µM, T = 37 °C, [Nile Red] = 1.25 µM. Comparing the HPLC degradation profiles with the disassembly rates of micelles, as was measured by fluorescence of Nile Red, greatly assisted in analyzing the correlation between the enzymatically induced degradation and the disassembly of the micelles. According to the HPLC data, hybrid 1 and 2 were degraded relatively fast in the presence of the enzyme and we observed the formation of partially cleaved intermediates, bearing one or two non-cleaved end-groups, respectively (Figures S61 and S62). Consequently the emission of Nile Red dropped in almost


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parallel with the disappearance of the parent hybrid. It is interesting to note that the decrease in fluorescence was greater for hybrid 1 than for hybrid 2. We attribute this difference to the different degree of enzymatic hydrolysis, which in the case of hybrid 2, resulted in the formation of hybrids that still contain two hydrophobic end-groups, while in the case of hybrid 1, cleavage of two end-groups led to the formation of an intermediate that has only one hydrophobic endgroup left. The difference in the hydrophobicity of these partially cleaved hybrids can explain the higher residual fluorescence of Nile red in the case of hybrid 2. In contrast to hybrids 1 and 2, hybrids 3 and 4 demonstrated much higher tolerance towards the enzymatic hydrolysis under these conditions and only partial degradation of the parent hybrid was observed within 24 hours. In addition, their “early” partially cleaved intermediates should be significantly more hydrophobic than the ones of hybrids 1 and 2 and therefore could probably participate in the selfassembly of the micelles. This can explain the significantly slower decrease in Nile Red's emission over time in comparison with the degradation rates of the parent hybrids, which were directly measured by HPLC. Due to the higher tolerance of hybrids 3 and 4 towards PGA, and as hybrids 5 and 6 were expected to display even higher micellar stability, an additional set of measurements was conducted using a 50-fold increase in the concentration of PGA (from 0.1 µM to 5 µM) and a 2fold decrease in the concentration of the hybrids (from 160 µM to 80 µM). As described before, the degradation of the parent hybrids was monitored by HPLC, while the disassembly rates of the micelles were derived from time-dependent decrease in Nile Red's emission intensities. Under these conditions, the degradation of hybrids 3 and 4 occurred much faster compared to the first set of conditions and “early” partially cleaved intermediates were further degraded into “late” intermediates and to the fully hydrolyzed hybrid in the case of hybrid 4 (Figures 11 and S66,


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respectively). Due to the lower hydrophobicity of these “late” intermediates, they could be expected to completely disassemble and as a result, the decrease in Nile Red's emission, indicative of the disassembly of the micelles, correlated well with the consumption of the parent hybrids (Figure 12). Hybrid 5, which had a total of 8 end-groups, formed even more stable micelles and degraded slower under the same conditions. Interestingly, hybrid 6 displayed almost full resistance towards the high dose of PGA and its degradation over a period of 5 days was negligible (Figures 12 and S68).

Figure 11. HPLC overlay for the enzymatic degradation of hybrid 3. [Hybrid] = 80 µM, [PGA] = 5 µM, T = 37 °C, λ = 260 nm.


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Figure 12. Enzymatic degradation of hybrids 3-6 monitored by a) HPLC and b) Emission intensity of Nile Red at 630 nm. [Hybrid] = 80 µM, [PGA] = 5 µM, T = 37 °C, [Nile Red] = 1.25 µM. In addition to the combination of HPLC and Nile Red techniques, the disassembly of the micelles was also directly confirmed by DLS. Figure 13 shows the DLS data at different time points for the different hybrids. It is interesting to note that as long as the parent hybrids still existed in significant amounts in the tested solution, the corresponding peaks of the micelles were still observed in the DLS data. However, after the complete degradation of the parent hybrid was confirmed by HPLC, the DLS measurements showed disappearance of the corresponding peaks and the formation of peaks with smaller diameters that correspond to the disassembled partially hydrolyzed hybrids and the enzyme. These results further confirm that although the enzymatic degradation of the hybrids did not lead to fully hydrolyzed hybrids, the partially formed hybrids that were formed are indeed more hydrophilic and do not form micelles under the given conditions.


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Figure 13. DLS measurements of a) hybrids 1-4 (160 µM in phosphate buffer, pH 7.4 with 0.1 µM PGA) and b) 3 - 6 (80 µM in phosphate buffer, pH 7.4 with 5 µM PGA) before addition of the enzyme (solid lines), 4 hours (dashed lines) and 48 hours (dotted lines) after incubation with PGA at 37 °C. The observed partial cleavage is in good correlation with our previous results for hybrid 2,27 which indicated the accumulation of a doubly cleaved hybrid that was confirmed by MALDITOF MS. The HPLC analysis allowed unprecedented ability to determine the molecular species in the studied solutions and helped us to reveal that in general, more end-groups could be cleaved when they are presented further away from each other. This trend indicates on an interference of the charged amines, which are formed after enzymatic hydrolysis, with further enzymatic


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degradation of the adjacent hydrophobic substrate. Hence, in general, branching of the dendron through allyl-containing branching units led to higher degree of hydrolysis, regardless of the actual rate of disassembly as illustrated by comparing hybrids 3 and 4 (Figures 11 and S66, respectively). To ensure that the partial degradation did not resulted from a lost of activity of the enzyme due to the prolong incubation in the buffer solution at 37 °C, we synthesized a low molecular weight substrate of PGA43 and used it to test the enzymatic activity after incubation of the enzyme in the buffer solution for 24 hours. The observed hydrolysis rate was found to be similar to the enzymatic hydrolysis rate for a freshly made solution of the enzyme (Figure S69), demonstrating that the enzyme did not lose its activity during the incubation time. Although the partial cleavage of the amphiphilic hybrids 1 – 6 did not affect the ability of their micelles to disassemble, the molecular origin of the interference with the full cleavage is the subject of future molecular modeling studies of these highly tunable polymeric amphiphiles. It is extremely intriguing to consider the relatively small changes in molecular weight between the hybrids and the drastic degree of change in micellar stability upon increasing the number of end-groups. In our previously reported work,27 we studied hybrids with different PEG lengths (2 kDa, 5 kDa and 10 kDa) and the same dendron, and while 10 kDa based hybrids disassembled in less than two hours, the 5 kDa and 2 kDa based hybrids disassembled in roughly two and a half hours, and four hours, respectively ([hybrid] = 160 µM; [PGA] = 0.14 µM) . Hence we were fascinated to see that in the current work, although the differences in the molecular weights of the hydrophobic dendrons were significantly subtler than between the different PEG lengths, the rate differences were remarkably larger and span through a much wider range of stabilities towards the enzymatic degradation. These results highlight the critical role that the polydispersity of the hydrophobic block may play in tuning the enzymatically triggered


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disassembly rates of polymeric amphiphiles.31 As small changes in the molecular weight of the hydrophobic part seem to result in significant changes in the degradation rates, one may question the ability to precisely adjust the disassembly rates of polymeric amphiphiles due to the inherited polydispersity of the lipophilic block. It is interesting to estimate that a 1:1 molar mixture of hybrids 1 (Mn = 6 kDa) and 6 (Mn = 8.1 kDa) would have a number average molecular weight of 7.05 kDa and PDI of 1.02 (based on a the theoretical molecular weights), which is actually lower than the PDI of the PEG block itself. It is then fascinating to note that such a mixture, which will be considered to be of very low polydispersity, will actually contain two species with extremely different stabilities towards enzymatic degradation. The observed sensitivity of the micellar stability towards minor differences in the hydrophobic part, which emerges from the polydisperse nature of most polymers, may help to explain the many examples of partial enzymatic degradation of amphiphilic polymers, which have been reported in the scientific literature.24,44–47 Summary and conclusions A series of six amphiphilic enzyme-responsive PEG-dendron hybrids was prepared using an extremely modular and high-yielding synthetic approach. The synthesized hybrids selfassembled into spherical nano-sized polymeric micelles in aqueous media and disassembled once incubated in the presence of a chosen enzyme. In addition, the ability of the micelles to encapsulate small hydrophobic guests within their cores and to release them was demonstrated using the solvatochromic Nile Red dye. Utilization of dendritic scaffolds as the responsive blocks allowed ultimate control over the number of enzymatically-cleavable end-groups, which was found to greatly affect the obtained micellar stability of the self-assembled monomers towards enzymatic degradation. The high molecular precision enabled us to study, in a step-wise


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fashion, the massive differences in micellar stabilities that could be gained by relatively small structural changes of the dendron-based blocks. The wide range of stabilities was shown to span from highly sensitive to practically non-degradable assemblies according to the number of endgroups, demonstrating the high tunability of the disassembly rates and their strong correlation with the molecular structure of the polymeric hybrids. This modular and facile approach will hopefully inspire the fabrication of future enzyme-responsive polymeric platforms with adjustable degradation and disassembly rates for various biomedical applications. Furthermore, this work highlights the key role of hydrophobicity in controlling the accessibility of the cleavable groups to the activating enzymes, and the tremendous effect of minor changes in the hydrophobic block and its polydispersity on the stability of assembled structures towards enzymatic degradation. ASSOCIATED CONTENT Detailed experimental information, characterization data and control experiments. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Roey J. Amir, Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT


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This research was supported by the Israel Science Foundation (Grants No. 966/14 and 2221/14). R.B. acknowledges the support of Israel Science Foundation (Grant No. 550/15). REFERENCES (1)

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(34) Whitton, G.; Gillies, E. R. J. Polym. Sci. Part A Polym. Chem. 2015, 53, 148. (35) Sharma, R.; Naresh, K.; Chabre, Y. M.; Rej, R.; Saadeh, N. K.; Roy, R. Polym. Chem. 2014, 5, 4321. (36) Abbassi, L.; Chabre, Y. M.; Kottari, N.; Arnold, A. A.; André, S.; Josserand, J.; Gabius, H.-J.; Roy, R. Polym. Chem. 2015, 6, 7666. (37) Jayakumar, K. N.; Bharathi, P.; Thayumanavan, S. Org. Lett. 2004, 6, 2547. (38) Killops, K. L.; Campos, L. M.; Hawker, C. J. J. Am. Chem. Soc. 2008, 130, 5062. (39) Hoogenboom, R. Angew. Chemie - Int. Ed. 2010, 49, 3415. (40) Lowe, A. B. Polymer (Guildf). 2014, 55, 5517. (41) Gillies, E. R.; Jonsson, T. B.; Fréchet, J. M. J. J. Am. Chem. Soc. 2004, 126, 11936. (42) Chandel, A. K.; Rao, L. V.; Narasu, M. L.; Singh, O. V. Enzyme Microb. Technol. 2008, 42, 199. (43) Weinstain, R.; Baran, P. S.; Shabat, D. Bioconjug. Chem. 2009, 20, 1783. (44) Amir, R. J.; Zhong, S.; Pochan, D. J.; Hawker, C. J. J. Am. Chem. Soc. 2009, 131, 13949. (45) Samarajeewa, S.; Shrestha, R.; Li, Y.; Wooley, K. L. J. Am. Chem. Soc. 2012, 134, 1235. (46) Samarajeewa, S.; Zentay, R. P.; Jhurry, N. D.; Li, A.; Seetho, K.; Zou, J.; Wooley, K. L. Chem. Commun. (Camb). 2014, 50, 968. (47) Tucker, B. S.; Getchell, S. G.; Hill, M. R.; Sumerlin, B. S. Polym. Chem. 2015, 6, 4258.


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SYNOPSIS. Precise adjustment of the number of enzymatically cleavable hydrophobic end-groups through highly modular synthetic approach allows access to polymeric hybrids with broad range of micellar stabilities.


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Modular Synthetic Approach for Adjusting the ... - ACS Publications

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