Reversible dimerization of polymeric amphiphiles acts as a molecular

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Reversible dimerization of polymeric amphiphiles acts as a molecular switch of enzymatic degradability Ido Rosenbaum, Ram Avinery, Assaf J Harnoy, Gadi Slor, Einat Tirosh, Uri Hananel, Roy Beck, and Roey J. Amir Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01150 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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Reversible dimerization of polymeric amphiphiles acts as a molecular switch of enzymatic degradability Ido Rosenbaum,†,§ Ram Avinery,∥,§ Assaf J. Harnoy,†,§ Gadi Slor,†,§ Einat Tirosh,‡,§ Uri Hananel,‡,§ Roy Beck,∥,§ and Roey J. Amir*,†,§,# †Department of Organic Chemistry, School of Chemistry, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 6997801, Israel §Tel Aviv University Center for Nanoscience and Nanotechnology, Tel-Aviv University, TelAviv 6997801, Israel School of Physics and Astronomy, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv

∥

6997801, Israel ‡Department of Physical Chemistry, School of Chemistry, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 6997801, Israel #

BLAVATNIK CENTER for Drug Discovery, Tel-Aviv University, Tel-Aviv 6997801, Israel

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

ACS Paragon Plus Environment

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ABSTRACT Enzyme-responsive polymeric micelles have great potential as drug delivery systems due to the high selectivity and overexpression of disease-associated enzymes, which could be utilized to trigger the release of active drugs only at the target site. We previously demonstrated that enzymatic degradation rates of amphiphilic PEG-dendron hybrids could be precisely tuned by gradually increasing the hydrophobic to hydrophilic ratio. However, with the increase in hydrophobicity, the micelles rapidly became too stable and could not be degraded, as often encountered for many other amphiphilic assemblies. Here we address the challenge to balance between stability and reactivity of enzymatically degradable assemblies by utilizing reversible dimerization of di-block polymeric amphiphiles to yield jemini amphiphiles. This molecular transformation serves as a tool to control the critical micelle concentration of the amphiphiles in order to tune their micellar stability and enzymatic degradability. To demonstrate this approach, we show that simple dimerization of two polymeric amphiphiles through a single reversible disulfide bond significantly increased the stability of their micellar assemblies toward enzymatic degradation, although the hydrophilic to hydrophobic ratio was not changed. Reduction of the disulfide bond led to de-dimerization of the polymeric hybrids and allowed their degradation by the activating enzyme. The generality of the approach is demonstrated by designing both esterase- and amidase-responsive micellar systems. This new molecular design can serve as a simple tool to increase the stability of polymeric micelles without impairing their enzymatic degradability.


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INTRODUCTION Polymeric nano-assemblies such as micelles have gained increasing interest in the past few decades due to their ability to serve as nano-carriers for the encapsulation of hydrophobic drugs and diagnostic probes.1–3 It is clear that such polymeric delivery system must be highly stable in order to avoid premature off-target release of their molecular cargo. The systems must also have a controlled release mechanism that will allow the selective release of their payload at the target site.4,5 Although various types of stimuli-responsive assemblies based on amphiphilic block copolymers that can respond to light,6,7 reduction,8–10 or changes in pH11–13 or temperature14,15 have been explored as potential delivery systems, the utilization of enzyme-responsive micelles offers great advantages.16 The over-expression of various enzymes at different sites of diseased tissues, such as the overexpression of cathepsin B17,18 and matrix metalloproteins (MMPs)19,20 in different types of tumors have been well documented, offering the potential to utilize these enhanced enzymatic activities to selectively trigger the release of the molecular payload of enzyme-responsive assemblies.21,22 Furthermore, even in the case of passive accumulation of polymeric assemblies at tumors due to the enhanced permeability and retention effect,23–26 ideally the polymeric carriers should be biodegradable to facilitate their excretion from the body after completing their role. Hence, designing polymeric assemblies with controllable enzymatic degradation rates is a major goal in the field of drug delivery systems. Various degradable polymers, such as polyesters, have been explored to address this goal.27–29 However, unlike “dimensionless” stimuli (i.e., light or temperature) or low molecular weight types of stimuli (e.g., changes in pH or the presence of reducing agents), which can easily diffuse into the micelles and alter the chemical or physical properties of the hydrophobic blocks that are collapsed inside the core of the micelles,


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enzymes cannot penetrate into the core of the micelles due to their comparable size range).30 In works reported by the groups of Heise31,32 and Thayumanavan33–36 the observed kinetic trends point strongly toward equilibrium-based enzymatic degradation that occurs through the equilibrium between the micelles and free monomers in the solution.37 It is very likely that these monomers are significantly more accessible to the enzyme, which can hydrolyze them and increase their hydrophilicity, ultimately leading to the disassembly of the micelles. Recently we reported on amphiphilic PEG-dendrons bearing enzymatically cleavable hydrophobic end-groups.38,39 Cleavage of the lipophilic end-groups by an amidase38 or an esterase39 led to a significant increase in the hydrophilicity of the hydrolyzed polymeric hybrids and to the disassembly of the micelles. PEG-dendron hybrids, which were initially introduced by Frechet, Gitsov, Hawker, and Wooley in the early 1990s,40 offer a higher degree of molecular precision in comparison to their linear analogs as the number of functional groups can be precisely tuned by the generation of the dendron and the multiplicities of its core and branching units.41 Taking advantage of the high structural precision of these polymeric hybrids, we gradually increased the number42 and/or length43 of the hydrophobic end-groups in order to tune enzymatic degradation rates. However, both for amidase and esterase responsive hybrids, above a certain degree of hydrophobicity, the micellar structures became too stable to be hydrolyzed even after long incubation with the activating enzymes.42,43 This limited degradation results from the low degree of exchange between the micellar and monomeric forms. Poor enzymatic degradation has been frequently encountered for many other amphiphilic block copolymers containing degradable hydrophobic blocks, demonstrating the need to develop new strategies to achieve both micellar stability and controlled enzymatic degradability.15,36,44–46


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Herein we demonstrate that dimerization of polymeric amphiphiles can serve as a generic molecular approach that enables design of polymeric amphiphiles with high stability against enzymatic degradation that can regain their enzymatic degradability by de-dimerization (Figure 1). The polymeric amphiphiles were based on amphiphilic PEG-dendrons with a thiol group at their focal point, enabling the reversible dimerization of the hybrids. To illustrate the generality of this approach, we studied the responses of the micelles to both an esterase and an amidase by adjusting the type of dendritic end-groups.

Figure 1. Schematic representation of dimerization of amphiphilic PEG-dendron hybrids bearing enzymatically cleavable dendritic end-groups (in red) to form non-degradable micellar nanocarriers. De-dimerization of the amphiphilic hybrids regenerates their capability to be degraded by the activating enzyme, leading to the formation of hydrophilic hybrids and disassembly of the micelles.


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MOLECULAR DESIGN Amphiphilic PEG-dendron hybrids with two types of enzymatically responsive dendrons were designed (Scheme 1): a dendron bearing four 4-methoxybenzoate end-groups (hybrid 1), which can serve as substrates for porcine liver esterase (PLE)47 and a dendron with four phenylacetamide end-groups (hybrid 2) that can be cleaved by penicillin G amidase (PGA).48 Both hybrids were functionalized with a thiol moiety; this allowed reversible dimerization of the hybrids by oxidation of the thiols or reduction of the disulfide bond, respectively. In addition to the thiol-functionalized hybrids 1 and 2, we also designed two hybrids that had methyl thio-ether moieties instead of the thiols and the same esterase or amidase cleavable groups (hybrids 1a and 2a, respectively). These two hybrids, which lack the thiol moiety and hence the ability to dimerize, served as controls and their micellar stability and response to the enzymatic degradation were compared to those of hybrids 1 and 2 after de-dimerization.


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Scheme 1. Structures of PEG-dendron hybrids 1, 1a, 2, and 2a. EXPERIMENTAL SECTION Materials: Poly (Ethylene Glycol) methyl ether 5kDa, 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%), Penicillin G Amidase from Escherichia coli (PGA), Esterase from porcine liver (PLE), Allyl

bromide

(99%),

N,N'-dicyclohexylcarbodiimide

(DCC,

99%),

N,N′-

Diisopropylcarbodiimide (DIC), 1-Hydroxybenzotriazole hydrate (HOBT), Fmoc-Lys(Boc)-OH (98%), 2-(Boc amino)ethanethiol, 4-Methoxybenzoic acid, 3-mercaptopropionic acid,


3-

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(methylthio)propionic acid and Sephadex® LH20 were purchased from Sigma-Aldrich. Cystamine hydrochloride (98%) and DIPEA were purchased from Merck. O-(Benzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 99.9%)

was purchased from

Chem-Impex. Trifluoroacetic acid (TFA) and 2-Mercaptoethanol (99%) were purchased from Alfa Aesar and phenyl acetic acid was purchased from Fluka. 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, Inc.

Synthesis of Hybrids: Hybrid 7. 150 mg (25.8 µmol) of Hybrid 649 were dissolved in DMF:DCM (2 mL 3:1 v/v). 4Methoxybenzoic acid (516 µmol), DCC (516 µmol) and DMAP (catalytic) were added and the reaction was allowed to stir at room temperature overnight. The crude mixture was loaded as is on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained white solid was dried under high vacuum. (146 mg, 90% yield). 1H-NMR (CDCl3): δ 7.95 (t, J = 7.9 Hz, 8H, arom H), 7.05 (s, 1H, NH-CO-Ar) 7.05 (t, J = 2.2 Hz, 2H, arom H), 6.86 (d, J = 7.0 Hz, 8H, arom H),6.74 (t, J = 5.9 Hz, -NH-CO-CH2-), 6.60 (t, J = 2.2 Hz, 1H, arom H), 4.56 (t, 1H, J = 2.4, NH-Boc), 4.28-4.45 (m, 9H,S-CH2-CH2-O- + CO-CH-NH-), 4.164.26 (m, 4H, arom-O-CH2), 3.48-3.82 (m, 530H, PEG backbone), 3.36 (s, 3H, OMe), 3.23-3.28 (m, 2H, -CH-S-), 2.55-3.09 (m, 16H, CH2-S-), 1.71-1.84 (m, 3H,O-CH2-CH2-CH2- + Lys-Hβ) 1.38-1.49 (m, 13H, Boc-NH-CH2-CH2-CH2-CH2-CH + Boc + Lys-Hβ);


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C-NMR (CDCl3) δ

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171.1, 166.4, 165.64, 165.60, 163.03, 163.01, 159.1, 135.6, 131.2, 121.8, 121.7, 113.2, 105.8,71.5, 70.1, 69.7, 69.3, 68.9, 63.5, 63.2, 58.6, 54.9, 53.1, 45.2, 38.2, 34.5, 31.5, 31.2, 31.1, 30.2, 29.2, 27.9, 27.8, 22.4, 22.2, 13.7 ; FT-IR, ν(cm-1): 2885, 1709, 1652, 1592,1540, 1466, 1359, 1341, 1279, 1240, 1146, 1100, 1060, 960, 842; GPC: Mn = 6.2kDa, ĐM = 1.04. Expected Mn = 6.3kDa. Hybrid 1-Trt. 64 mg (10 µmol) of hybrid 7 were deprotected with TFA in DCM (2 mL, 1:1 v/v). The crude was evaporated and then re-dissolved in DMF (1.5 mL). 3-(methylthio)propionic acid (50 µmol), HBTU (50 µmol) and DIPEA (150 µmol) were added and the reaction was stirred at room temperature overnight. The crude was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained white solid was dried under high vacuum (63 mg, quantitative yield).1H-NMR (CDCl3): δ 7.95 (t, J = 7.9 Hz, 8H, arom H),7.17-7.39 (m, 15H, Trt H), 7.13 (d, J = 7.21 Hz,1H, NH-CO-Ar), 7.03 (t, J = 2.2 Hz, 2H, arom H), 6.88 (d, J = 7.0 Hz, 8H, arom H), 6.77 (t, J = 5.9 Hz, NH-CO-CH-), 6.63 (t, J = 2.2 Hz, 1H, arom H), 5.61 (t, J = 5.9 Hz, NH-CO-CH2-), 4.42-4.55 (m, 9H,S-CH2CH2-O- + CO-CH-NH-), 4.17-4.29 (m, 4H, arom-O-CH2), 3.45-3.83 (m, 594H, PEG backbone), 3.38 (s, 3H, OMe), 3.26-3.31 (m, 2H, -CH-S-), 2.56-3.06 (m, 18H, CH2-S-), 2.41-2-45 (t, J = 7.3 Hz, 2H, -CO-CH2-CH2-S-), 1.74-1.83 (m, 3H,O-CH2-CH2-CH2- + Lys-Hβ) 1.33-1.51 (m, 5H NH-CH2-CH2-CH2-CH2-CH + Lys-Hβ);

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C-NMR (CDCl3) δ 166.5, 165.6, 163.03,163.02,

159.1,144.2, 131.2, 129.1, 127.5, 126.3, 121. 8,121.7, 113.2, 105.9, 71.5, 70.1, 69.7, 69.3, 68.9, 66.3, 63.5, 63.2, 58.6, 54.9, 53.1, 45.2, 38.2, 35.1, 34.5, 31.5, 31.2, 31.1, 30.2, 29.2, 28.5, 27.8,


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27.3; FT-IR, ν(cm-1): 2885, 1710, 1606, 1512, 1466, 1359, 1341, 1278, 1241, 1146, 1100, 1060, 960, 842; GPC: Mn = 6.5kDa, ĐM = 1.02. Expected Mn = 6.4kDa. Hybrid 1. The Trityl of compound 1-Trt (60 mg) was deprotected by dissolving in DCM:TFA mixture for 30 minutes (2 mL, 1:1 v/v) and Et3SiH was added (10 µmol) to the reaction.Solvents were evaporated to dryness and the crude mixture was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained white solid was dried under high vacuum (63 mg, quantitative yield). The de-protection was confirmed by 1H-NMR and HPLC. GPC: Mn = 6.6kDa, ĐM = 1.02. Expected Mn = 6.4kDa. Dimerization: Oxidation was performed by purging oxygen into solution of hybrid 1 (160µM in water) for 12 hours. GPC: Mn = 13.6kDa, ĐM = 1.07. Expected Mn = 12.9kDa; MALDI-TOF MS: molecular ion of the dimer centered at 12.8kDa. Hybrid 1a. 70 mg (11.1 µmol) of Hybrid 7 were deprotected by TFA in DCM (2 mL, 1:1 v/v). The crude was evaporated and then re-dissolved in DMF (1.5 mL). 3-(methylthio)propionic acid (55 µmol), HBTU (55 µmol) and DIPEA (221 µmol) were added and the reaction was stirred at room temperature overnight. The crude was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained white solid was dried under high vacuum (64 mg, 91% yield). 1H-NMR (CDCl3): δ 7.97 (t, J = 7.9 Hz, 8H, arom H), 7.18 (d,


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J =7.21 Hz, 1H, NH-CO-Ar), 7.05 (t, J = 2.2 Hz, 2H, arom H), 6.88 (d, J = 7.0 Hz, 8H, arom H), 6.79 (t, J = 5.9 Hz, NH-CO-CH-), 6.64 (t, J = 2.2 Hz, 1H, arom H), 6.06 (t, J = 5.9 Hz, NH-COCH2-), 4.43-4.59 (m, 9H,S-CH2-CH2-O- + CO-CH-NH-), 4.19-4.29 (m, 4H, arom-O-CH2), 3.483.82 (m, 484H, PEG backbone), 3.38 (s, 3H, OMe), 3.24-3.31 (m, 4H, -CH-S- + -CH2-CH2-NH), 2.57-3.09 (m, 18H, CH2-S-), 2.39-2-43 (t, J = 7.3 Hz, 2H, -CO-CH2-CH2-S-), 2.05 (s, 3H, SMe), 1.81-1.86 (m, 3H,O-CH2-CH2-CH2- + Lys-Hβ) 1.39-1.54 (m, 5H -NH-CH2-CH2-CH2-CH2CH + Lys-Hβ);

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C-NMR (CDCl3) δ 171.1, 166.6, 165.64, 165.60,163.03, 159.1, 131.2, 121.8,

121.7, 113.2, 105.9,71.5, 70.1, 69.7, 69.3, 68.9,63.5, 63.2, 58.6, 54.9, 53.1, 45.2, 38.2, 37.01, 35.9, 34.5, 31.5, 31.2, 31.1, 30.2, 29.5, 29.2, 28.4, 27.8, 22.2, 15.2; FT-IR, ν(cm-1): 2884, 1709, 1606, 1512, 1466, 1359, 1341, 1279, 1241, 1147, 1100, 1060, 960, 842; GPC: Mn = 6.5kDa, ĐM= 1.02. Expected Mn = 6.4kDa; MALDI-TOF MS: molecular ion centered at 6.4kDa. Dendron 9. 820 mg (3.36 mmol) of compound 850 were dissolved in ACN (5mL per 1g). 2(Boc amino)ethanethiol (8eq.) and 2,2-Dimethoxy-2-phenylacetophenone (DMPA, 0.08eq.) were added. The solution was purged with nitrogen for 15 minutes and then placed under UV light at 365nm for 2 hours. ACN was evaporated to dryness and the crude was purified by silica column (Hexane:EtOAc 50:50). Solvetns were evaporated to dryness and the off-white solid was dried under high vacuum. The product was obtained as an off-white solid (2.54 g, 79% yield). 1HNMR (CDCl3): δ 7.16 (d, J = 2.2 Hz, 2H, arom H) 6.66 (t, J = 2.2 Hz, 1H, arom H), 5.18 (s, 2H,NH-CO-), 5.11 (s, 2H-NH-CO-), 4.16-4.20 (m, 4H, arom-O-CH2-), 3.87 (s, 3H, O-CH3) 3.29-3.32 (m, 8H, CH2-NH-Boc), 3.12-3.15 (m, 2H, -CH-S-), 2.65-2.93 (m, 12H, CH2-S-), 1.23 (s, 36H, Boc);

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C-NMR (CDCl3) δ 166.6, 159.4, 155.9, 132.2, 108.3, 106.8, 79.5, 69.8, 52.3,

45.1, 40.5, 40.1, 34.4, 33.02, 32.09, 28.5; FT-IR: ν(cm-1): 2976, 2362, 1753, 1640, 1595, 1547,


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1502, 1479, 1413, 1390, 1365, 1326, 1301,1248, 1158, 1046, 949, 864, 768; HRMS (ESI) calculated for C42H72N4O12S4 975.3922 (MH+), found 975.3927. Dendron 10. 930 mg (1.05 mmol) of compound 9 were dissolved in 15 mL DCM:TFA (1:1 v/v). Solvents were evaporated to dryness and then re-dissolved in DCM (10 mL). 4-Nitrophenol 2 –phenyl acetate (10.5 mmol) and DIPEA (18.9 mmol) were added and the reaction stirred overnight at RT. The crude mixture was evaporated and purified by silica column (Hexane:MeOH 90:10). The fractions that contained the product were unified and the solvents were evaporated to dryness and the off-white solid was dried under high vacuum. The product was obtained as an off-white solid (875 mg, 88% yield). 1H-NMR (CDCl3): δ 7.17-7.26 (m, 20H, CH2-Ar-H), 7.16 (d, J = 2.2 Hz, 2H, arom H) 6.64 (t, J = 2.2 Hz, 1H, arom H), 6.49 (t, J = 5.8 Hz, 2H-NH-CO-), 6.41 (t, J = 5.8 Hz, 2H-NH-CO-), 4.16-4.20 (m, 4H, arom-O-CH2-), 3.88 (s, 3H, O-ME), 3.48 (s, 8H, CH2-Ar) 3.32-3.37 (m, 8H, CH2-NH-Boc), 3.02-3.07 (m, 2H, -CH-S-), 2.58-2.81 (m, 12H, CH2-S);

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C-NMR (CDCl3) δ 171.38, 171.33, 166.6, 159.4, 134.9, 132.2,

129.3, 128.9, 127.3, 108.3, 106.8, 69.8, 52.4, 44.7, 43.6, 39.3, 39.1, 34.2, 32.2, 31.2; FT-IR: ν(cm-1): 3071, 2361, 1772, 1636, 1588, 1541, 1507, 1473, 1419, 1397, 1381,1323, 1070, 956, 769; HRMS (ESI) calculated for C54H64N4O8S4 1025.3682 (MH+), found 1025.3685. Dendron 11. 764 mg (0.745 mmol) of compound 10 were dissolved in MeOH:Dioxane (10 mL 3:1 v/v) and 0.5 mL of NaOH 4N was added. After 6 hours HCl 1M was added until pH was 2. The organic solvents were evaporated and then re-dissolved in EtOAc (70mL) was washed with HCl 1M (70mL) followed by Brine (3x50mL). The solution was dried over MgSO4 and then evaporated to dryness and the off-white solid was dried under high vacuum. The product was obtained as an off-white solid (750 mg, quantitative yield). 1H-NMR (CDCl3): δ 7.2-7.32 (m, 20H, CH2-Ar-H), 7.19 (d, J = 2.2 Hz, 2H, arom H) 6.63 (t, J = 2.2 Hz, 1H, arom H), 6.39 (t,


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J = 5.8 Hz, 2H-NH-CO-), 6.30 (t, J = 5.8 Hz, 2H-NH-CO-), 4.05-4.13 (m, 4H, arom-O-CH2), 3.52 (s, 8H, CH2-Ar) 3.34-3.42 (m, 8H, CH2-NH-Boc), 3.03-3.09 (m, 2H, -CH-S-), 2.58-2.84 (m, 12H, CH2-S); 13C-NMR (CDCl3) δ 171.79, 171.74, 166.6, 159.4, 134.9, 132.2, 129.6, 129.1, 127.5, 108.7, 106.8, 69.9, 44.8, 43.7, 39.5, 39.2, 34.4, 32.4, 31.4; FT-IR: ν(cm-1): 2976, 2361, 1725, 1710, 1692, 1659, 1585, 1549, 1530, 1482, 1468, 1217; HRMS (ESI) calculated for C53H62N4O8S4 1011.3527 (MH+), found 1011.3529. Hybrid 13. 225 mg (42.7 µmol) of Hybrid 1249 were dissolved in DMF:DCM (2mL 3:1 v/v). Compound 11 (171 µmol), DIC (171 µmol), DIPEA (171 µmol) and HOBT (171 µmol) were added and the reaction was put in microwave initiator for 1 hour at 60 0C. The crude mixture was loaded as is on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained white solid was dried under high vacuum. (224 mg, 84% yield). 1HNMR (CDCl3): δ 7.59 (s, 1H, NH-CO-Ar) 7.2-7.33 (m, 20H, CH2-Ar-H), 7.1 (d, J = 2.2 Hz, 2H, arom H), 6.89 (t, J = 5.9 Hz, NH-CO-CH-) 6.59 (t, J = 2.2 Hz, 1H, arom H), 6.28 (t, J = 5.8 Hz, 2H-NH-CO-), 6.21 (t, J = 5.8 Hz, 2H-NH-CO-), 4.71 (t, 1H, J = 2.4,NH-Boc), 4.57 (m, 1H, CO-CH-NH-), 4.08-4.15 (m, 4H, arom-O-CH2), 3.45-3.82 (m, 542H, PEG backbone), 3.37-3.42 (m, 11H, CH2-Ar + OMe), 3.05-3.12 (m, 4H, -CH-S- + CH2-NH-Boc), 2.55-2.85 (m, 16H, CH2S-), 1.40-1.44 (m, 13H, Boc-NH-CH2-CH2-CH2-CH2-CH + Boc);

13

C-NMR (CDCl3) δ 171.87,

171.52, 171.39, 167.2, 159.5, 134.9, 129.5, 129.1, 127.4, 106.7, 105.2, 72.1, 70.7, 69.9, 69.6, 63.8, 59.1, 53.9, 44.85, 44.82, 43.8, 39.5, 39.2, 38.9, 34.3, 32.4, 31.7, 31.4, 29.8, 28.6, 23.2; FT-


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IR, ν(cm-1): 2930, 2853, 1711, 1657, 1564, 1524, 1451, 1338, 1274, 1135, 1101, 994, 891, 833, 754; GPC: Mn = 6.2kDa, ĐM = 1.02. Expected Mn = 6.3kDa. Hybrid 2-Trt. 57 mg (9.1 µmol) of hybrid 13 were deprotected with dissolving in DCM:TFA mixture for 30 minutes (2 mL, 1:1 v/v). The crude solvents were evaporated and then redissolved in DMF (1.5 mL). 3-(Tritylthio)propionic acid (45.7 µmol), HBTU (45.7 µmol) and DIPEA (137 µmol) were added and the reaction was stirred at room temperature overnight. The crude mixture was loaded as is on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1mL) followed by addition of Hexane (3mL). DCM and Hexane were evaporated to dryness and the obtained white solid was dried under high vacuum (55 mg, 92% yield). 1H-NMR (CDCl3): δ 7.59 (t, J = 5,1H, NH-CO-Ar) 7.18-7.41 (m, 29H, Trt + CH2-Ar-H), 7.1 (d, J = 2.2 Hz, 2H, arom H), 6.89 (t, J = 5.9 Hz, NH-CO-CH-) 6.59 (t, J = 2.2 Hz, 1H, arom H), 6.28 (t, J = 5.8 Hz, 2H, -NH-CO-), 6.21 (t, J = 5.8 Hz, 2H, -NH-CO-), 4.52 (m, 1H, -CO-CH-NH-), 4.08-4.17 (m, 4H, arom-O-CH2), 3.45-3.81 (m, 624H, PEG backbone), 3.35-3.38 (m, 11H, CH2-Ar + OMe), 3.05-3.15 (m, 4H, -CH-S- + -CH2-CH2-NH-), 2.53-2.81 (m, 18H, CH2-S-), 2.41-2.44 (t, J = 7.2 Hz, 2H, -CO-CH2-CH2-S-), 1.76-1.83 (m, 3H,O-CH2CH2-CH2- + Lys-Hβ) 1.35-1.48 (m, 5H -NH-CH2-CH2-CH2-CH2-CH + Lys-Hβ);

13

C-NMR

(CDCl3) δ 171.3, 170.93, 170.91, 170.83, 170.68, 166.7, 158.9, 144.2, 134.4, 129.1, 128.9, 128.5, 127.5, 126.8, 126.2, 106.1, 104.7, 71.5, 71.3, 69.9, 69.3, 69.1, 58.6, 44.3, 43.2, 38.9, 38.6, 38.4, 38.3, 35.1, 33.7, 31.8, 31.04, 30.8, 29.23, 29.18, 28.5, 27.9, 27.3, 22.4 ; FT-IR, ν(cm-1): 2885, 1642, 1536, 1467, 1359, 1342, 1279, 1240, 1146, 1103, 960, 842 ;GPC: Mn = 6.3kDa, ĐM = 1.02. Expected Mn = 6.5kDa.


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Hybrid 2. The Trityl of compound 2-Trt (50 mg) was deprotected with dissolving in DCM:TFA mixture for 30 minutes (2 mL, 1:1 v/v) and Et3SiH was added (9.1 µmol) to the reaction. The crude was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained white solid was dried under high vacuum (50 mg, quantitative yield). The de-protection was confirmed by 1H-NMR and HPLC.GPC: Mn = 6.4kDa, ĐM= 1.05. Expected Mn = 6.4kDa. Dimerization: Oxidation was performed by purging oxygen into solution of hybrid 2 (160µM in water) for 12 hours. GPC: Mn = 12.8kDa, ĐM = 1.06. Expected Mn = 12.7kDa; MALDI-TOF MS: molecular ion of the dimer centered at 12.7kDa. Hybrid 2a. 40 mg (6.4 µmol) of Hybrid 13 were deprotected with dissolving in DCM:TFA mixture for 30 minutes (2 mL, 1:1 v/v).The crude was evaporated and then re-dissolved in DMF (1.5 mL). 3-(methylthio)propionic acid (32 µmol), HBTU (32 µmol) and DIPEA (96 µmol) were added and the reaction was stirred at room temperature overnight. The crude was loaded on a MeOH based LH20 SEC column. The fractions that contained the product were unified and the MeOH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeOH and solidification of the product, the oily residue was re-dissolved in DCM (1 mL) followed by addition of Hexane (3 mL). DCM and Hexane were evaporated to dryness and the obtained white solid was dried under high vacuum (37 mg, 93% yield). 1H-NMR (CDCl3): δ 7.61 (s,1H, NH-CO-Ar) 7.20-7.31 (m, 20H, CH2-Ar-H), 7.07 (d, J = 2.2 Hz, 2H, arom H), 6.9 (t, J = 5.9 Hz, NH-CO-CH-) 6.59 (t, J = 2.2 Hz, 1H, arom H), 6.32 (t, J = 5.8 Hz, 2H, -NH-CO-),


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6.2 (t, J = 5.5 Hz, 2H, -NH-CO-), 4.55 (m, 1H, -CO-CH-NH-), 4.1-4.16 (m, 4H, arom-O-CH2), 3.45-3.81 (m, 614H, PEG backbone), 3.35-3.38 (m, 11H, CH2-Ar + OMe), 3.03-3.24 (m, 4H, CH-S- + -CH2-CH2-NH-), 2.35-2.81 (m, 20H, CH2-S- + -CO-CH2-CH2-S), 2.05(s, 3H, S-Me), 1.77-1.83 (m, 3H,O-CH2-CH2-CH2- + Lys-Hβ) 1.39-1.54 (m,5H-NH-CH2-CH2-CH2-CH2-CH + Lys-Hβ); 13C-NMR(CDCl3)δ 171.3, 171.02, 170.91, 170.89, 170.81, 166.7, 158.9, 135.6, 134.4, 128.9, 128.5, 126.8, 106.1, 104.7, 71.5, 71.3, 70.1, 69.3, 69.1, 58.5, 53.3, 44.3, 43.2, 38.9, 38.6, 38.29, 38.28, 35.9, 33.7, 31.8, 31.04, 30.8, 29.5, 29.19, 28.4, 27.8, 27.3, 22.3, 22.2, 15.8, 13.6 ; FT-IR, ν(cm-1): 2885, 1587, 1548, 1467, 1450, 1342, 1279, 1100, 960, 842; GPC: Mn = 6.6kDa, ĐM =1.03. Expected Mn = 6.4kDa. ; MALDI-TOF MS: molecular ion centered at 6.4kDa. RESULTS AND DISCUSSION Synthesis of the polymeric hybrids. To introduce the crosslinking group, we synthesized PEG-lysine (3), bearing two orthogonal protecting groups (Boc and Fmoc), which could be orthogonally deprotected in order to allow sequential conjugation of the dendron and the thiol moiety. Esterase-responsive hybrids were synthesized on the PEG following a previously reported divergent approach:49 The Fmoc moiety from 3 was removed, and the deprotected amine was coupled with a di-yne-based branching unit 4 by amidation to yield hybrid 5. The triple bonds were then reacted through a thiol-yne reaction51–54 with 2-mercaptoethanol yielding tetra-hydroxy hybrid 6.49 Esterification with 4-methoxy benzoic acid resulted in hybrid 7 (Scheme 2).


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Scheme 2. Divergent synthesis of esterase-responsive hybrid 7. Hybrids 2 and 2a, which were designed to have amide end-groups, were synthesized through a convergent approach based on synthesis of the dendron “off “the PEG (Scheme 3). Branching unit 8 was reacted with Boc-protected cysteamine to yield dendron 9, followed by deprotection of the Boc group and amidation with 4-nitrophenyl 2-phenyl-acetate to yield dendron 10. Next, the carboxylate group at the focal point of the dendron was hydrolyzed, and the formed acidfunctionalized dendron, 11 was conjugated to the Boc-protected PEG-lysine (12) to yield hybrid 13. The reason for the different synthetic approach is the lack of orthogonally of the Boc protecting groups that are used during the synthesis of the dendron block and the Boc protecting group of the PEG-lysine.


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Scheme 3. Convergent synthesis of amidase-responsive hybrid 13. To introduce the thiol functionalities, hybrids 7 and 13 were deprotected using TFA to remove the Boc group from the ε-amine of the Lys and allow its conjugation with 3(tritylmercapto)propionic acid followed by deprotection of the trityl group to yield hybrids 1 and 2, respectively. Hybrids 1a and 2a were prepared similarly by conjugation with 3(methylmercapto)propionic acid after the deprotection of the Boc group (Scheme 4). The hybrids and dendrons were characterized by 1H- and 13C-NMR, IR, mass spectroscopy, UV, and HPLC, and the experimental results confirmed the synthesis of the desired hybrids (see SI).


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Scheme 4. Functionalization of hybrids 7 and 13 to yield hybrids 1, 1a, 2, and 2a. After completing the syntheses of hybrid 1 and 2, we dimerized them by purging aqueous solutions of the self-assembled polymers with oxygen. Gel permeation chromatography (GPC) analysis of the dimerization reaction indicated a high degree (~90%) of dimer formation for both hybrids 1 and 2 (Figure 2). The reversibility of the dimerization was then evaluated by reduction of the disulfide bond with 0.1 M dithiothreitol (DTT) and complete de-dimerization and regeneration of the monomeric hybrids was observed (Figure 2).


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Table 1. Molecular properties of dimers of hybrids 1 and 2, hybrids 1a and 2a, and their micellar assemblies. Hybrid

Mna (kDa)

ĐM a

Mpb Calc. (kDa) Mnc (Da)

PEG dendron wt. ratioc

CMCd (µM; mg/L)

RHe (nm)

Hybrid

13.6

1.07

12.8

12,855

78:22

0.5 ± 0.2

8 ± 1 6.4 ± 0.1

1 dimer

(6.6)g

(1.02)g

(6,427)g

(78:22)g 7 ± 3

Hybrid

6.5

1.02

6,441

78:22

6.4

4±1

Rgf (nm)

Agg. Num.f

17h (dimers)

7 ± 1 5.8 ± 0.1

30h

9 ± 1 6.7 ± 0.1

10 h

26 ± 6

1a Hybrid

12.8

1.06

2 dimer

(6.4)g

(1.05)g

Hybrid

6.6

1.03

12.7

6.4

12,718

78:22

5±2

(6,359)g

(78:22)g 60 ± 25

6,373

78:22

21 ± 2

(dimers) 7 ± 1 5.7 ± 0.1

19 h

130 ± 15

2a a

Molar-mass dispersity, ĐM measured by GPC using PEG standards. b Molecular weight of the highest peak (Mp) measured by MALDI-TOF mass spectroscopy. c The calculated molecular weights (Mn) and PEG:dendron weight ratios were calculated based on PEG 5 kDa and the calculated exact mass of the dendrons. d Critical micelle concentration (CMC) determined using Nile red. e Hydrodynamic radius (RH) measured by dynamic light scattering (DLS). f Radius of gyration (Rg) and aggregation numbers measured by SAXS. g Measured before dimerization. h Error estimate for scattering intensity (I0) is produced by “GNOM” and may be propagated to aggregation numbers, but the dominant uncertainty, in solute concentration, was not considered and may be on the order of few percent.


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Figure 2. GPC traces of the hybrids (a) 1 and (b) 2 before (black line) and after (blue line) oxidation and after reduction by DTT (red line). Self-assembly of the polymeric hybrids and their response to reduction. After the reversibility of the dimerization was confirmed by GPC, we studied the self-assembly of the polymers into micelles and their response to DTT. First, we determined the critical micelle concentration (CMC) of the two dimers of hybrids 1 and 2, and their non-dimerizing analogues, 1a and 2a, by using the solvatochromic dye Nile red.55 These measurements gave the first indication for the higher stability of the dimerized hybrids, as their CMCs were significantly lower than the values for the non-dimerizing hybrids 1a and 2a (Table 1). Next, dynamic light scattering (DLS) was used to measure the sizes of the self-assembled structures (Figure 3) and diameters of 16 ± 1 nm and 17 ± 1 nm were observed for the dimerized hybrids 1 and 2, respectively. Interestingly, smaller sizes were obtained for hybrids 1a and 2a, indirectly


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indicating on the effect of dimerization on the packing of the micelles. DLS measurements of hybrids 1 and 2 in the presence of DTT showed that the reduced de-dimerized hybrids gave micelles of similar diameters as the non-dimerizing hybrids 1a and 2a. Transmission electron microscopy (TEM) images (Figures S27 and S28), further confirmed the formation spherical assemblies and increased sizes for the dimeric-based systems in comparison with the nondimerizing ones.

Figure 3. DLS data for dimerized hybrids (a) 1 and (b) 2 and their non-dimerizing analogues 1a and 2a (solid red lines), respectively. Dimerized hybrids in buffer (solid green lines), in the presence of 0.1 M DTT (dotted black lines), incubated with the enzyme (dashed purple lines), and in the presence of both DTT and the enzyme (solid blue lines) were analyzed. X-axes are presented in log scale.


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To further study these changes in the diameters of the micellar assemblies and to get information on the aggregation numbers, we used the small-angle X-ray scattering (SAXS) technique. We were encouraged to see that the SAXS results showed that smaller sizes were measured for the assemblies of hybrids 1a and 2a in comparison with the micellar assemblies of dimerized hybrids 1 and 2 (Figure 4 and Table 1). Using SAXS, we also estimated the masses of the polymers in the micelles from the forward scattering intensity, I0.43 The aggregation numbers in Table 1 refer to the number of hybrids/dimers in each micelle and were calculated by dividing the measured micelle weight by the molecular weight of the specific hybrid/dimer. The difference in aggregation number between hybrids 2 and 2a was smaller than the relative variance in the sizes of their assemblies, and may be attributed to the more oval shape of the micelles of dimerized hybrid 2. These results clearly indicate that the dimerization affects the molecular packing of the self-assembled structures, although the hydrophilic to hydrophobic ratio is not changed upon dimerization of the hybrids.

Figure 4. Pair-distance distributions of dimerized hybrids 1 (solid red line) and 2 (solid black line) and their non-dimerizing analogues 1a (dashed red line) and 2a (dashed black line). The distributions were produced by fitting the SAXS measurements using indirect Fourier transform as described in the SI.


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Next we examined how the redox-responsive changes in both the molecular structures of the hybrids and the sizes of their assemblies will affect the loading capacities of the micelles. To address this question, we studied the changes in fluorescence intensity of encapsulated Nile red molecules. We assumed that if the ability of the micelles of hybrids 1 and 2 to encapsulate the solvatochromic dye were impaired upon de-dimerization, a significant decrease in the fluorescence intensity would be observed as the Nile red molecules will be released into the aqueous environment. Fluorescence spectra of hybrids 1 and 2 before and after the addition of DTT showed only minor decrease in fluorescence intensity (Figures S30 and S32), indicating that the micelles retained their loading capacities after the de-dimerization of the hybrids and the subsequent decrease in size of the micelles. Enzyme-responsive degradation and disassembly. The next step after completing the characterization of the assembled micelles and their redox-response was to evaluate their enzymatic degradation. Based on the significantly lower CMC values of the dimerized hybrids, we expected the micellar assemblies of the dimers to be substantially more stable than micelles that are composed of the reduced hybrids or the non-dimerizing hybrids (1a and 2a). To follow the enzymatic disassembly, we measured the fluorescence intensity of Nile red-containing micelles in the presence of the activating enzyme. As the enzymatic degradation increases the overall hydrophilicity of the hybrids, the polymeric micelles are expected to disassemble. This should force the Nile red molecules to migrate to the more polar aqueous environment, leading to a decrease in their fluorescence intensity. Hence following the change in fluorescence allowed us to indirectly quantify micelle stability in the tested solutions. Time-dependent fluorescence spectra of Nile red for dimerized hybrids 1 and 2 in the presence of the activating enzymes


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(Figure 5a, b) were measured and the obtained kinetic data was compared to the non-dimerizing hybrids 1a and 2a (Figure 5c, d). The micelles of the non-dimerizing hybrids readily disassembled in the presence of the corresponding activating enzymes as indicated by the decrease in fluorescence intensity. In contrast, only minor decreases in fluorescent intensities were observed in solution of the micelles of dimerized hybrids 1 and 2 in the presence of enzyme (Figure 5c, d). When micelles of hybrids 1 and 2 were incubated with DTT, disassembly rates in the presence of enzyme were similar to those of the non-dimerizing hybrids 1a and 2a (Figure 5c, d). These experiments demonstrated the potential of dimerized hybrids to form highly stable micelles, which upon reduction and de-dimerization, regained their enzymatic responsiveness. In parallel with the fluorescence measurements, HPLC was used to directly follow the degradation of the parent hybrids and formation of enzymatically degraded hybrids and the cleaved end-groups. As expected, almost no degradation of the dimers in the presence of the activating enzymes was observed by HPLC (Figure S34 and S37), confirming the high stability of the dimerized hybrids 1 and 2 toward enzymatic degradation. When the esterase-responsive hybrid 1 was incubated in the presence of both DTT and an esterase, we observed a clear transformation of the dimer into monomeric form and a significant increase in its degradation rate (Figures 5c and S33); this rate was very similar to the enzymatic degradation rate that was obtained for hybrid 1a (Figure 5e). Furthermore, the HPLC chromatograms revealed that the enzymatic degradation resulted in fully hydrolyzed hybrids. The HPLC degradation rate of hybrid 1 and the decrease in fluorescence intensity, which is indicative of the presence of micelles, showed an excellent correlation (Figure 5e). When hybrids 2 and 2a were studied, we noticed that these hybrids were not fully hydrolyzed by the enzyme (Figures S36 and S38). We previously reported similar partial degradation for


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other amidase-responsive hybrids and attributed it to interference of the positively charged ammonium end-groups that are formed after the enzymatic hydrolysis with enzyme activity.42 Although the hybrids were not fully degraded, we observed good correlation between the HPLC and the fluorescence data (Figure 5f), confirming the enzymatic degradation of the parent hybrid and the disassembly of the micelles, respectively. To get further support for the enzymatically-induced disassembly of the micelles we studied samples also by DLS. The obtained DLS data for the degraded hybrids clearly showed the disappearance of the larger micellar assemblies and the formation of smaller structures corresponding to the degraded hydrophilic hybrids (Figure 3). In contrast, micelles of dimerized hybrids 1 and 2 showed no change in their size upon addition of the enzyme, further indicating that the dimers based micelles are resistant to enzymatic degradation. The reported results clearly demonstrate that simple dimerization of the polymeric amphiphilic hybrids caused a significant change in their packing and in micellar stability toward enzymatic degradation as evident from the changes in micelle size, CMC values, and degradation rates for both the esterase- and amidase-responsive hybrids. Interestingly, the de-dimerization through reduction of the di-sulfide bond did not caused the disassembly of the micelles as often reported for redox-responsive assemblies.56 It is important to note that experimental and theoretical studies of low molecular weight gemini-surfactants have demonstrated the stability of assemblies due to the unique two head/two tail structure.57 Taking advantage of differences between the monomeric and dimeric forms of the polymeric amphiphiles, we were able to almost completely inhibit the enzymatic degradation of the hybrids and then “turn on” their degradability by a simple de-dimerization induced by reduction.

This approach opens the way for design of

polymeric assemblies that will combine high stability and full enzymatic degradability, which is


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often challenging to achieve for highly stable micelles. Furthermore, this approach will allow the design of delivery platforms with high selectivity, as the activation of the release of micellar payload will require the presence of two different types of stimuli, making these systems to function as an AND logic gate.33 We are currently investigating the ability to expand the presented molecular approach to other types of reversible dimerizations through pH- and photoresponsive bonds that will allow de-dimerization by changes in pH or irradiation with light.

Figure 5. Time-dependent fluorescence spectra in the presence of the activating enzyme and DTT for dimers of (a) hybrid 1 and (b) hybrid 2. Change in maximum emission intensity of Nile


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red for (c) hybrid 1 and (d) hybrid 2 in the presence of the activating enzyme (red line), DTT (blue line), and both DTT and the enzyme (green line) in comparison with the data for hybrid 1a and hybrid 2s (dashed black lines). Comparison of the degradation of the hybrids (determined by HPLC, black line) and the change in fluorescence emission of encapsulated Nile red (green line) for (e) hybrid 1 and (f) hybrid 2 in the presence of both DTT and the enzyme. The HPLC degradation of hybrid 1a and hybrid 2a (red lines) are presented for comparison. The amount of the first intermediate of the hybrid 2 cleavage is plotted in orange in panel f. CONCLUSIONS To summarize, taking advantage of the high molecular precision of PEG-dendron hybrids, we prepared both esterase- and amidase-responsive polymeric amphiphiles that self-assembled into micellar structures. Introduction of a single thiol group at the focal point of the hybrids allowed their dimerization through the formation of a single disulfide bond. The formed dimers had significantly lower CMC values than the non-dimerizing hybrids and extremely high stability toward enzymatic degradation. Reduction of the disulfide bonds led to de-dimerization of the hybrids, which regained their enzymatic degradability. The presented results demonstrate the potential of reversible dimerization to serve as a simple and efficient molecular tool to rationally increase the resistance of polymeric assemblies to enzymatic degradation and dilution while maintaining the ability of the micelles to undergo complete enzymatically induced disassembly after de-dimerization of the polymeric building blocks. This molecular approach is also envisioned to increase the selectivity of such delivery platforms as they will require the presence of two types of stimuli in order to trigger the disassembly, serving as an AND logic gate.


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ASSOCIATED CONTENT Supporting Information. Detailed experimental information, characterization data, CMC measurements, UV and fluorescence spectra, SAXS data, HPLC conditions and data, encapsulation and release 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] Present Addresses †If an author’s address is different than the one given in the affiliation line, this information may be included here. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT 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 550/15) REFERENCES (1)

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SYNOPSIS Reversible dimerization of the amphiphilic hybrids can serve as a simple molecular approach form highly stable micellar assemblies. De-dimerization of the amphiphiles turns on their enzymatic degradability.


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Reversible dimerization of polymeric amphiphiles acts as a molecular

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