Supramolecular Linear-g-Hyperbranched Graft Polymers - American

Article pubs.acs.org/Macromolecules

Supramolecular Linear‑g‑Hyperbranched Graft Polymers: Topology and Binding Strength of Hyperbranched Side Chains Christian Moers,†,‡ Lutz Nuhn,† Marcel Wissel,§ René Stangenberg,§ Mihail Mondeshki,∥ Elena Berger-Nicoletti,† Anja Thomas,† David Schaeffel,§ Kaloian Koynov,§ Markus Klapper,§ Rudolf Zentel,† and Holger Frey*,† †

Institute of Organic Chemistry, Johannes Gutenberg-University Mainz (JGU), Duesbergweg 10-14, D-55128 Mainz, Germany Graduate School Materials Science in Mainz (MAINZ), Staudinger Weg 9, D-55128 Mainz, Germany § Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany ∥ Institute of Inorganic Chemistry and Analytical Chemistry, Johannes Gutenberg-University Mainz (JGU), Duesbergweg 10-14, D-55128 Mainz, Germany ‡

S Supporting Information *

ABSTRACT: Complex, reversible hyperbranched graft polymer topologies have been obtained by spontaneous self-assembly. Well-defined adamantyl- and β-cyclodextrin-functionalized polymers were employed to generate linear-g-(linear−hyperbranched) supramolecular graft terpolymers. For this purpose the synthesis of monoadamantyl-functionalized linear polyglycerols (AdalinPG) and hyperbranched polyglycerols (Ada-hbPG) as well as poly(ethylene glycol)-block-linear polyglycerol (Ada-PEG-blinPG) and poly(ethylene glycol)-block-hyperbranched poly(glycerol) (Ada-PEG-b-hbPG) block copolymers was established. Isothermal titration calorimetry (ITC) with β-cyclodextrin revealed a shielding effect of hyperbranched polyglycerol for the adamantyl functionality, which was significantly less pronounced when using a linear spacer chain between the adamantyl residue and the hyperbranched polyglycerol block. Additionally, welldefined poly(2-hydroxypropylamide) (PHPMA) with pendant β-cyclodextrin moieties was synthesized via RAFT polymerization and sequential postpolymerization modification. Upon mixing of the β-cyclodextrin-functionalized PHPMA with Ada-PEG-bhbPG, a supramolecular linear-g-(linear−hyperbranched) graft terpolymer was formed. The self-assembly was proven by ITC, diffusion-ordered NMR spectroscopy (DOSY), and fluorescence correlation spectroscopy (FCS).

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INTRODUCTION Cyclodextrins (CD) are a class of cyclic oligosaccharides that are finding multiple applications, e.g., as pharmaceutical solubilizers,1 in CD-mediated polymerizations,2 in cosmetics,3 and in agricultural industry.4 Because of their torus-like shape providing a hydrophilic exterior and a hydrophobic cavity, they are capable of incorporation and dissolution of smaller hydrophobic guest molecules. β-CD, for example, encloses adamantane and its derivatives with generally high association constants in aqueous solution.5 The high binding energy of βCD and adamantane as the driving force for the supramolecular assembly of complex architectures in solution has recently attracted broad attention.6 Even for stabilization of advanced drug delivery systems carrying highly sensitive, biological active compounds (e.g., siRNA), polyplex systems involving β-CD/ adamantane assemblies show high efficacy for both delivery7 and degradation.8 Various macromolecular structures ranging from linear block copolymers9−14 to miktoarm star polymers15−17 have been assembled and investigated. Combining hyperbranched polyglycerol (hbPG) grafted from β-CD and an adamantyl-functionalized alkyl chain, a linear−hyperbranched supramolecular amphiphile was prepared.18 However, to date, © 2013 American Chemical Society

supramolecular graft structures include only two examples. Bertrand et al. synthesized comb-shaped polymers based on a β-CD grafted methacrylate backbone in combination with linear adamantyl-functionalized poly(acrylic acid) side chains.19 Zang and co-workers prepared dendronized polymers consisting of a β-CD-functionalized polymethacrylate backbone and adamantyl-functionalized oligo(ethylene glycol)-based first- and second-generation dendrons.20 Compared to dendrimers, hyperbranched polymers offer a much simpler synthetic protocol, while still exhibiting many features of dendrimers such as a high number of functional groups and a globular shape.21,22 Hyperbranched polyglycerol (hbPG) can be prepared in a facile one-step synthesis while still providing good molecular weight control and low to moderate polydispersity indices (PDIs) (Mw/Mn ≤ 1.9).23 Interestingly, hyperbranched polyglycerol structures have gained further attraction due to their high biocompatibility,24,25 enabling applications in the field of polymer-based therapeutics or Received: October 9, 2013 Revised: November 10, 2013 Published: December 3, 2013 9544

dx.doi.org/10.1021/ma402081h | Macromolecules 2013, 46, 9544−9553

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diagnostics.23,26,27 Among different macromolecular structures containing hyperbranched polymers, linear−hyperbranched block copolymers currently represent an emerging topic.28,29 On the other hand, to date, linear−hyperbranched graft copolymers have only been synthesized and studied in few works.30−35 While Pargen et al.33 used the macromonomer approach, Hsieh and co-workers grafted hyperbranched poly(ethylenimine) onto poly(allylamine).32 Recently, our group presented the first application of a graf ting-to strategy to obtain linear poly(2-hydroxypropyl methacrylamide) (PHPMA) polymers with highly biocompatible24 hyperbranched polyglycerol branched side chains.35 However, to the best of our knowledge, no linear-g-(linear−hyperbranched) graft terpolymers have been synthesized and investigated. In the case of the analogous perfect dendrimers, a major disadvantage in the graf ting-to strategy applied to the synthesis of dendronized polymers is the decreased reactivity of dendrons of higher generation, mainly caused by the shielding effect of the branched structure to its focal reactive group.36−38 This issue may be overcome by the introduction of a spacer moiety between the focal reactive group and the use of a less densely packed hyperbranched structure.

polymers11 while linear HPMA polymers with pendant CD units have not been reported.46 Mixing these linear β-CDfunctionalized PHPMA polymers with mono adamantylfunctionalized linear−hyperbranched poly(ethylene glycol)hyperbranched polyglycerol block copolymers provides access to a novel supramolecular linear-graft-(linear−hyperbranched) polymer architecture (Figure 1). To evaluate the accessibility of the focal adamantyl unit in the poly(ethylene glycol)-blockhyperbranched-polyglycerol copolymers, we have also synthesized hyperbranched polyglycerols with an adamantyl unit at the focal point without the PEG spacer. Comparison of the association constants of these polymers with β-CD (monomolecular or attached to an HPMA polymer backbone) permits an evaluation of the shielding effect of the hyperbranched polymer and the influence of the linear poly(ethylene glycol) (PEG) spacer by isothermal titration calorimetry (ITC).

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EXPERIMENTAL SECTION

All chemicals were purchased from Sigma-Aldrich, Acros Organics, and TCI and used as received, unless otherwise stated. Pentafluorophenol was obtained from Fluorochem, and Oregon Green Cadaverine was purchased from Invitrogen. Anhydrous tetrahydrofuran (THF) and 1,4-dioxane were freshly distilled from sodium or a sodium/potassium mixture, respectively. Anhydrous dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich and stored over activated molecular sieves (4 Å). 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized from diethyl ether and stored at −7 °C. Deuterated solvents were purchased from Deutero. β-CD used for isothermal titration experiments was dried over Sicapent (Merck) in vacuo prior to use. Ethoxyethyl glycidyl ether was synthesized as described in the literature.47 Hyperbranched polyglycerol (hbPG86) was prepared via the macroinitiator approach according to the literature.48 Potassium naphthalide was prepared as a stock solution (0.5 M) in anhydrous THF under argon atmosphere from equivalent amounts of potassium and freshly sublimated naphthalene. Preparative size exclusion chromatography (SEC) was carried out using Sephadex G25 and Millipore water as an eluent. Dialysis was either performed using Cellu Sep T1 membranes with molecular weight cutoff of 3500 g/mol (for Ada-hbPG162) or Spectra/Por 3 membranes with molecular weight cutoff of 6000−8000 g/mol (for P(cyclodextrin 10 -co-PA 7 -coHPMA58)) obtained from Carl Roth GmbH & Co. KG (Germany). Spectra/Por Biotech membranes with molecular weight cutoff of 100− 500 g/mol (for Ada-PEG13-b-hbPG35) and Cellu Sep H1 membranes with molecular weight cutoff of 1000 g/mol (for Ada-PEG13-b-hbPG72 and Ada-PEG13-b-hbPG107) were purchased from Bioron GmbH (Germany). 1 H and 13C NMR spectra were recorded at 300 and 75 MHz, respectively, using a Bruker AC300 or at 400 and 100 MHz, respectively, on a Bruker Avance-II 400 NMR spectrometer. The spectra were referenced internally to residual proton signals of the deuterated solvent used. 19F NMR spectra were recorded on a Bruker 400 MHz FT NMR spectrometer. The 2D 1H nuclear Overhouser effect spectroscopy (NOESY) spectra were recorded on a Bruker Avance DRX 400 NMR spectrometer. Diffusion ordered spectroscopy (DOSY) experiments were performed at 298 K in D2O at a typical concentration of 10 mg/mL on a Bruker Avance DRX 400 NMR spectrometer. Additional information concerning NOESY and DOSY NMR experiments is included in the Supporting Information. Size exclusion chromatography (SEC) measurements were performed in dimethylformamide (DMF), THF, or hexafluoroisopropanol (HFIP). Details can be found in the Supporting Information. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on a Shimadzu Axima CFR MALDI-TOF mass spectrometer using potassium trifluoroacetate as cationizing agent and dithranol (1,8,9-trihydroxyanthracene) or CHCA (α-cyano-4-hydroxycinnamic acid) as matrix.

Figure 1. Schematic representation of the formation of the supramolecular linear-g-(linear−hyperbranched) graft terpolymer.

In this work we present the synthesis of a β-CDfunctionalized linear PHPMA backbone and linear−hyperbranched poly(ethylene glycol)-block-hyperbranched polyglycerol block copolymers bearing a single adamantyl group at the focal point. PHPMA was chosen as a linear backbone due to the possibility to introduce multiple functionalities by preparation via the reactive ester approach.35,39−42 PHPMA is highly water-soluble and biocompatible39,43,44 thus rendering it a perfect support for pendant β-CD residues. However, PHPMA-CD conjugates only include star polymers based on a β-CD core45 and chain end β CD-functionalized PHPMA 9545


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Scheme 1. Synthetic Strategy for the Synthesis of Monoadamantyl-Functionalized Linear and Hyperbranched Polyglycerols (m = 0, Ada-linPG and Ada-hbPG, respectively) and Monoadamantyl-Functionalized Poly(ethylene glycol)-block-Linear Polyglycerol (Ada-PEG-b-linPg) and Poly(ethylene glycol)-block-Hyperbranched Polyglycerol (Ada-PEG-b-hbPG) Block Copolymers

stirring bar P(PFMA)75 (50 mg; 2.60 μmol of polymer or 195 μmol of reactive ester) was dissolved in anhydrous dioxane (2 mL) under an argon atmosphere. Oregon Green Cadaverine (387.8 μL of a 2.5 mg/ mL solution in DMSO; 1.95 μmol), propargylamine (3.75 μL, 58.6 μmol), and triethylamine (81.4 μL, 586 μmol) were added. The tube was immersed in an oil bath at 50 °C under vigorous stirring. After 16 h, a small sample was taken for 19F NMR analysis showing partial conversion of the pentafluorophenyl reactive ester. For complete conversion, 2-hydroxypropylamine (76.4 μL, 976 μmol) was added, and the reaction mixture kept stirring at 50 °C under an argon atmosphere. After an additional 24 h, 19F NMR analysis of a small sample of the reaction mixture revealed complete conversion of the reactive ester. Because of covalent attachment of the fluorescent dye, the resulting polymer could be isolated by size exclusion chromatography (SEC) with Sephadex G25 and Millipore water as eluent. All yellow-green fractions of high molecular weight were combined and lyophilized, affording P(PA17-co-HMPA58) as a voluminous orange powder. Yields: 22.4 mg (2.11 μmol), 81%. SEC (HFIP, PMMA-Std.): Mn = 23 000 g/mol; Mw = 29 000 g/mol; PDI = 1.26 − Mn(1H NMR, calc) = 10 600 g/mol). 1H NMR (300 MHz, DMSO-d6): δ [ppm] = 7.70−6.86 (br, 1H, −NH−); 4.90−4.40 (br, 1H, −OH); 3.88−3.55 (br, 1H, −CH(OH)−, and 1H, −NH−CH2− CC); 3.11−2.66 (br, −NH−CH2−CH(OH)−); 2.10−0.44 (br, 8H, −CH2− and −CH3 polymer main chain and −CH3 side chain of HPMA and 6H, CH2− and −CH3 polymer main chain and −CC− CH side chain of PA). Conjugation of Mono-(6-azido-6-desoxy)-β-cyclodextrin to Oregon Green Cadaverine Labeled Poly(propargylmethacrylamide-co-2-Hydroxypropylmethacrylamide) P(cyclodextrin10-co-PA7-co-HPMA58) (14). In a Schlenk tube equipped with a stirring bar P(PA17-co-HPMA58) (21.4 mg; 2.0 μmol of polymer or 34.0 μmol of alkyne moieties) dissolved in 3 mL of DMSO was mixed with mono-(6-azido-6-desoxy)-β-cyclodextrin (104.0 mg, 89.6 μmol) dissolved in 2 mL of Millipore water. To degas

Fourier transform IR (FT-IR) spectra were recorded using a Thermo Scientific iS10 FT-IR spectrometer equipped with a diamond ATR unit. Isothermal titration calorimetry (ITC) measurements were performed with a Microcal VP-ITC titration microcalorimeter (MicroCal, Inc., Northhampton, MA).49 Fluorescence correlation spectroscopy (FCS) was performed using an Olympus IX70 microscopy/FluoView300 setup with a PicoQuant FCS Upgrade and a Olympus UPLSAPO 60×/1.2 W Corr. water immersion objective. Detailed information is given in the Supporting Information. Synthesis of (Adamant-1-yl)methyloxypoly(ethylene glycol)-block-Hyperbranched Polyglycerol or (Adamant-1-yl)methyloxy-Hyperbranched-Polyglycerol. (Adamant-1-yl)methyloxypoly(ethylene glycol)-block-hyperbranched-poly(glycerol) and (adamant-1-yl)methyloxy-hyperbranched-poly(glycerol) were synthesized according to a four-step procedure established previously for other initiators.50−52 A detailed description of the experimental procedures is presented in the Supporting Information. Mono-(6-O-(p-tolylsulfonyl))-β-cyclodextrin. The product was prepared as described by Petter et al.53 Detailed experimental procedure and analytical data are given in the Supporting Information. Mono-(6-azido-6-desoxy)-β-cyclodextrin. Mono-(6-azido-6desoxy)-β-cyclodextrin was prepared according to a slighty modified literature synthesis.54 Detailed experimental procedure and analytical data are given in the Supporting Information. Synthesis of Poly(pentafluorophenol methacrylate) P(PFPMA) (12). Synthesis of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, pentafluorophenol methacrylate, and P(PFPMA) via RAFT polymerization was performed as described recently.55 A detailed experimental procedure is provided in the Supporting Information. Synthesis of Oregon Green Cadaverine Labeled Poly(propargylmethacrylamide-co-2-Hydroxypropylmethacrylamide) P(PA17-co-HMPA58) (13). In a Schlenk tube equipped with a 9546


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the reaction mixture, two freeze−pump−thaw cycles were applied before copper(II) acetate (8.0 mg, 44.0 μmol) and ascorbic acid (40.0 mg, 227.1 μmol) could be added under an argon atmosphere. Again, two additional freeze−pump−thaw cycles were applied before the reaction tube could be immersed in an oil bath at 40 °C under vigorous stirring. After 16 h the reaction mixture was exposed to air and then dialyzed against Millipore water for several days (including frequent water exchange) and subsequent lyophilization affording P(cyclodextrin10-co-PA7-co-HPMA58) as a voluminous orange powder. Yields: 39.6 mg (2.11 μmol), 81%. SEC (HFIP, PMMA-Std.): Mn = 27 400 g/mol; Mw = 35 000 g/mol; PDI = 1.28 − Mn(1H NMR, calc) = 22 100 g/mol). 1H NMR (400 MHz, DMSO-d6): δ [ppm] = 8.20− 6.65 (br, 1H, −NH− HPMA/PA and 2H, triazole cyclodextrin); 6.14−5.43 (m, 14H, C2−OH, C3−OH); 5.05−4.80 (m, 7H, C1H, cyclodextrin); and 4.74−4.67 (1H, −OH, HPMA); 4.58−4.26 (m, 6H, C6−OH, cyclodextrin); 4.11−3.43 (m, 28H, C3H, C5H, C6H2 cyclodextrin, and 1H, −CH(OH)− HPMA, and 1H, -NH−CH2CC PA); 3.34−3.09 (m, 14H, C2H, C4H cyclodextrin); 3.21−2.61 (br, −NH−CH2− HPMA); 2.10−0.44 (br, 8H, −CH2− and −CH3 polymer main chain and −CH3 side chain of HPMA, and 6H, CH2− and −CH3 polymer main chain and −CC−CH side chain of PA, and 5H, CH2− and −CH3 polymer main chain of cyclodextrin) (peak assignment was done supported by COSY and HSQC spectra). 13C NMR (100 MHz, DMSO-d6): δ [ppm] = 176.9 (CO); 102.4−101.7 (C1, cyclodextrin); 81.5 (C2−OH, cyclodextrin); 73.1−71.1 (C3, C4, C5 cyclodextrin); 64.7 (−CH(OH)− HPMA); 59.9 (C6−OH cyclodextrin); 54.3 (Cquat. polymer main chain); 47.6 (−NH−CH2− HPMA); 44.6 (−CH2− polymer main chain); 21.6 (−CH3 HPMA); 18.1 (−CH3 polymer main chain) (peak assignment was done supported by COSY and HSQC spectra).

Table 1. Characterization Data of Ada-PEEGE8, Ada-linPG9, Ada-PEG15-b-PEEGE9, Ada-PEG13-b-linPG8, and All AdahbPG and Ada-PEG-b-hbPG Samples no.

compositiona

Mnb

Mnc

Mnd

PDIe

DBf

1 2 3 4 5 6 7 8 9 10 11

Ada-PEEGE8 Ada-linPG9 Ada-hbPG35 Ada-hbPG60 Ada-hbPG77 Ada-hbPG162 Ada-PEG15-b-PEEGE9 Ada-PEG13-b-linPG8 Ada-PEG13-b-hbPG35 Ada-PEG13-b-hbPG72 Ada-PEG13-b-hbPG107

1600 770 2000 3200 4200 8200 2100 1500 2800 4800 5800

1400 870 2700 4600 5900 12100 2200 1300 3400 6100 8600

1100 760 2000 2700 3200 5200 1200 970 2500 4500 5000

1.21 1.17 1.41 1.72 1.48 1.79 1.16 1.13 1.33 1.28 1.54

0g 0g 0.54 0.59 0.60 0.61 0g 0g 0.57 0.60 0.62

a

Calculated from 1H NMR data using the integrated ratios of the methylene group of the 1-adamantylmethanol initiator (−O−CH2−C) at 2.96 ppm and the polyether backbone. bTargeted molecular weight in g/mol (for entries 2 and 7: calculated from 1H NMR data of the corresponding protected PEEGE polymer). cCalculated from 1H NMR data in g/mol. dDetermined by SEC (DMF, PEG standard) in g/mol. eDetermined by SEC (DMF, PEG standard). fCalculated from 13 C inverse gated decoupled data. gLinear polymers by definition have a degree of branching of 0.

literature.57,58 The DB values range between 0.55 and 0.62 and are in good agreement with the literature. Figure 2 shows the

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RESULTS AND DISCUSSION A. Adamantyl-Functionalized hbPGs. Novel hbPGs with exactly one adamantyl moiety at the focal unit of the polymer and its derivatives with a PEG spacer between the adamantyl group and the hbPG block have been synthesized. While a synthetic strategy based on postpolymerization modification of multifunctional hbPGs leads to polymers with multiadamantyl residues and a random distribution,56 the use of a suitable monofunctional alcohol in several steps (Scheme 1) allows incorporation of exactly one specific residue.50,52 In our case, the use of 1-adamantylmethanol, which has to the best of our knowledge not yet been used as an initiator for oxyanionic ring-opening polymerizations, required modification of the reported deprotonation strategy.50−52 Deprotonation by cesium hydroxide and subsequent azeotropic removal of water in vacuo using benzene was not possible, since 1-adamantylmethanol is able to sublimate. Thus, a mixture of cesium 1adamantylmethylate and cesium hydroxide initiated the polymerization of the epoxides. Characterization via MALDITOF spectrometry revealed the existence of both the (adamant-1-yl)methyloxy- and hydroxy-initiated polymer species. In consequence, a different deprotonation strategy was developed. An excess of 1-adamantylmethanol was dissolved in anhydrous benzene (over molecular sieve) under an argon atmosphere and deprotonated using potassium naphthalide. Subsequently, to exclude potential traces of water, benzene was removed in vacuo and potassium 1-adamantylmethoxide (AdaMeOK) was dried at 90 °C in vacuo overnight and used as initiator for the ring-opening polymerization of EO and EEGE, respectively. After cleavage of the protective group and hypergrafting of glycidol, the desired polymers were obtained with good molecular weight control and PDIs below 1.8 (see Table 1). The degree of branching (DB) was calculated from inverse gated proton decoupled 13C NMR data as described in the

Figure 2. MALDI-TOF MS spectrum of Ada-PEEGE8 (1) (b) (matrix: dithranol) as well as the SEC traces of Ada-linPG9 (2) and all Ada-hbPG samples (3−6) (c) (RI signal; eluent: DMF).

MALDI-TOF spectrum of Ada-PEEGE8 as well as the SEC traces of the resulting polymers Ada-linPG9 and all Ada-hbPGs. At high elution volumes SEC is even capable of separating oligomers of Ada-linPG9, which explains additional modes visible in its trace. B. β-Cyclodextrin Grafted HPMA Polymer. We chose the postpolymerization modification of poly(pentafluorophenyl 9547


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Scheme 2. Synthetic Strategy for the Synthesis of P(cyclodextrin10-co-PA7-co-HPMA58) (14)

copy (FCS) (reactive ester conversion was monitored by 19F NMR; compare Supporting Information Figure S42). However, according to different solubility properties of the precursor units (on the one hand, P(PFPMA) is only soluble in nonpolar organic solvents; on the other hand, cyclodextrin is only soluble in polar solvents, e.g., water), direct aminolysis of the P(PFPMA) with monoamine-functionalized cyclodextrin was not possible. For this purpose we chose the copper-catalyzed azide alkyne cycloaddition of monoazide-functionalized cyclodextrin onto the alkyne-containing HPMA polymers as the conjugation of carbohydrates onto water-soluble methacrylamide polymers has already been reported successfully.60 In our case, the click ligation of cyclodextrin to PHPMA was proven by SEC via a shift to smaller elution volumes and NMR

methacrylate) (P(PFPMA)) via aminolysis using 2-hydroxypropylamine and propargylamine to establish alkyne functionalities at the methacrylamide backbone for subsequent coppercatalyzed azide−alkyne cycloaddition using monoazide-functionalized cyclodextrin (Scheme 2). The combination of these two polymer modification reactions has recently turned out to be a powerful tool for introducing even advanced functionalities (e.g., glycopeptides) onto HPMA homo and block copolymers.42 RAFT polymerization itself already offers versatile access to complex macromolecular architectures,59 while especially the reactive ester approach toward HPMA polymers enables introduction of additional functionalities,35,39−42 e.g., aminolysis by a monoamine-functionalized fluorescent dye (Oregon Green Cadaverine) for detection by fluorescent correlation spectros9548


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Figure 3. 1H NMR spectrum of P(cyclodextrin10-co-PA7-co-HPMA58) (14) (lower spectrum) obtained from P(PA17-co-HPMA58) (13) (upper spectrum) via copper-catalyzed azide alkyne cycloaddition with β-CD-azide.

spectroscopy by appearance of the triazole and β-CD signals, as demonstrated in Figures 3 and 4.

Figure 4. SEC traces of P(PA17-co-HPMA58) (13) (black, solid) and P(cyclodextrin10-co-PA7-co-HPMA58) (14) (red, dashed).

C. Characterization of Association by Isothermal Titration Calorimetry. It is an intriguing question whether a linear spacer unit between the hbPG and the complexed adamantyl unit is of importance for the accessibility of the adamantyl residue. To determine association constants between β-CD and adamantyl residues and to evaluate the shielding effect of the hyperbranched block, ITC experiments in water were performed. To a solution of pure β-CD in water a solution of adamantyl-functionalized polymer (compounds 2−6 and 8− 11, Table 1) was slowly added. All associations are enthalpy driven, and the stoichiometry is in accordance with a roughly equimolar association (with a maximum deviation of 0.3). A graphical representation of the calculated association constants in Figure 5 clearly demonstrates the shielding effect of hyperbranched polyglycerol on the association. After a strong decrease of the association constant compared to the linear precursor at lower molecular weight of the hyperbranched block, an additional increase in molecular weight does not lead to an enhanced effect. Instead, association constants seem to asymptotically approach a final value. Most probably, glycerol

Figure 5. Association constant vs molecular weight for Ada-PEG13-blinPG8 (8) (first red circle) and Ada-PEG13-b-hbPG samples (9−11) (red circles, top left image) as well as Ada-linPG9 (2) (first black square) and Ada-hbPG samples (3−6) (black squares, top right image), with respect to β-CD.

in the periphery at elevated molecular weight does not provide any additional shielding effect due to the high aqueous solubility of hbPG, its imperfect branching (DB 0.55−0.62, Table 1), and high flexibility of the polyether backbone and thus less densely packed structure. The effect of the PEG spacer separating the adamantyl function from the branched structure is obvious by studying the dependence of the association constant for the complex 9549


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PEG spacer, the shielding effect for adjacent β-CD units is even more pronounced In all three cases the stoichiometry of association shifts to lower values compared to the mixtures with free β-CD, thus indicating that fewer β-CD units are available. This is also in accordance with a restricted access to neighboring β-CD cavities upon inclusion complex formation. Transferred to the synthesis of covalently bound linear− hyperbranched graft copolymers via a graf ting-to strategy, one can conclude that the introduction of a spacer between the branched structure and the anchor group is an important prerequisite for high grafting densities. Nonetheless, the experiments clearly support formation of the anticipated linear-g-(linear−hyperbranched) graft terpolymer when mixing P(cyclodextrin10-co-PA7-co-HPMA58) and Ada-PEG13-b-hbPG72 or Ada-PEG13-b-hbPG107, respectively. By combining P(cyclodextrin10-co-PA7-co-HPMA58) and AdahbPG77, a supramolecular linear−hyperbranched graft copolymer was formed. D. Association of the Supramolecular Polymer Structure Probed by NMR Spectroscopy and FCS Experiments. Diffusion-ordered NMR spectroscopy (DOSY) has been frequently applied to the characterization of macromolecular supramolecular assemblies, e.g., supramolecular polymers in solution.62 Hasegawa et al. proved the formation of supramolecular polymers based on β-CD and adamantyl-functionalized dimers by diffusion NMR experiments,12 while Barner-Kowollik and co-workers used this technique to demonstrate the formation of H-shaped terpolymers.63 Examples for the characterization of supramolecular graft copolymers by DOSY were among others presented by Pan et al.64 as well as by Meijer and co-workers.65 In this study all DOSY experiments were conducted at total concentrations of 10 mg mL−1 of the interacting polymers. The reason is that the association constant between Ada-PEG13-bhbPG107 with β-CD attached to the linear HPMA polymer was found to be about 4.8 × 104 L mol−1; thus, the majority of the adamantyl-functionalized polymers would not be associated with the linear backbone at lower concentrations. In addition, the formation and dissociation of intermolecular complexes are fast on NMR time scale, and therefore the measured diffusion coefficient is a weighted average of all associated and dissociated species present in the mixture. Comparing the diffusion coefficients of the adamantyl residues of Ada-PEG13-bhbPG107 and the respective mixture of Ada-PEG13-b-hbPG107 and P(cyclodextrin)10-co-PA7-co-HPMA58), a shift in diffusion coefficient by a factor of 2 is revealed as shown in Figure 6. This effect mirrors the formation of a new supramolecular host− guest complex that exhibits an increased hydrodynamic radius Rh that translates to a lower diffusion coefficient (Table 3). Therefore, the observation of an increase in hydrodynamic radius from 1.4 to 2.7 nm in the case of Ada-PEG13-b-hbPG107 and from 3.5 to 4.8 nm when P(cyclodextrin10-co-PA7-coHPMA58) is considered, is in good agreement with the formation of the supramolecular polymer structure. Further confirmation for the formation of the supramolecular graft polymer is obtained by the nuclear Overhauser effect (NOESY) NMR spectra. NOESY is a two-dimensional NMR technique used to map nuclear Overhauser effect correlations between protons situated within 4 Å from one another and is generally well-suited to study host/guest complex formation.66−68 As depicted in Figure S47, new cross-correlation peaks appear upon mixing of P(cyclodextrin10-co-PA7-co-

formation between β-CD and the adamantyl residue on the polymer structure (Figure 5). For the linear−hyperbranched polymers the final association constant is still 75−80% of the macroinitiator’s value. On the other hand, the value decreases by 74% in the systems lacking the PEG spacer. One can conclude that polymers containing a PEG spacer provide much easier access to the adamantyl unit, which results in significantly higher association constants for these hyperbranched systems and a lower decrease in association constant from the linear precursor. When comparing our results to the adamantyl-cored oligo(ethylene glycol) dendrons of Yan et al.,61 the less densely packed structure of hyperbranched polymers is clearly reflected in their weaker shielding effect. To exclude the possibility of a contribution of nonspecific hbPG/β-CD interaction to the association, an ITC experiment with β-CD and hbPG86 without any adamantyl functionalities was also conducted. The heat flow observed does not differ from that of a simple dilution experiment (Supporting Information Figure S48a). Thus, no interaction whatsoever appears to take place between β-CD and unfunctionalized hbPG. To assess the influence of the attachment of β-CD to linear backbone, three adamantyl-functionalized polymers (5, 10, and 11, Table 1) were chosen for a detailed study with P(cyclodextrin10-co-PA7-co-HPMA58). The determined association constants are summarized in Table 2. Table 2. Association Constants and Numbers Obtained from ITC Experiments for Titration of Selected AdamantylFunctionalized Polymers to Host Polymer P(cyclodextrin10co-PA7-co-HPMA58) (14) guest polymer

K (L mol−1)

Ada-hbPG77 (5) Ada-PEG13-b-hbPG72 (10) Ada-PEG13-b-hbPG107 (11)

(3.0 ± 0.7) × 104 (5.5 ± 0.5) × 104 (4.8 ± 0.5) × 104

When P(cyclodextrin10-co-PA7-co-HPMA58) was titrated with Ada-PEG13-b-hbPG107 in an ITC experiment, significant differences between free β-CD (Figure 5) and β-CD attached to a polymer backbone (Table 2) were observed. The association constant decreases by more than a factor of 2 to 4.83 × 104 L mol−1. Because of the chosen synthetic pathway, β-CD units are distributed randomly along the HPMA polymer chain and their spatial distance varies. Bonding of one hyperbranched polymer chain may easily impede access to a neighboring β-CD unit completely or at least hinder association of another polymer chain even for remote β-cyclodextrin moieties. Additionally, the linear HPMA backbone may further have to unfold from random coil to allow association of several hyperbranched polymers. Similar results were obtained for the combination of Ada-PEG13-b-hbPG72 and P(cyclodextrin10-coPA7-co-HPMA58). To evaluate the influence of the PEG spacer by direct comparison of the two structures with and without PEG spacer, Ada-hbPG77 was chosen for the last experiment as its molecular mass is similar to Ada-PEG13-b-hbPG72. When titrating AdahbPG77 to P(cyclodextrin10-co-PA7-co-HPMA58), the observed association constant is only 2.98 × 104 L mol−1. This value is lower than the one for the association with free β-CD and also lower compared to the association of Ada-PEG13-b-hbPG72 and P(cyclodextrin10-co-PA7-co-HPMA58). Because of the lack of a 9550


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For the next experiments a PBS-based stock solution of P(cyclodextrin 10 -co-PA 7 -co-HPMA 58 ) and Ada-PEG 13 -bhbPG107 at concentrations of c = 3 mg mL−1 (0.13 mM) for P(cyclodextrin10-co-PA7-co-HPMA58) and c = 13 mg mL−1 (1.5 mM) for Ada-PEG13-b-hbPG107 was prepared. When diluting this mixture with a 1.5 mM solution of Ada-PEG13-b-hbPG107 in PBS, FCS experiments revealed an increase in hydrodynamic radius of the labeled P(cyclodextrin10-co-PA7-co-HPMA58) compound to 4.9 nm. This high excess of the adamantylfunctionalized compound is necessary to counteract the strong dilution of the Oregon Green labeled β-CD grafted PHPMA and to lead to detectable association at its given association constant. The observed decrease in diffusion coefficient and thus the increase in hydrodynamic radius to 4.9 nm corresponds to the anticipated formation of the linear-g(linear−hyperbranched) graft terpolymer. Additionally, hydrodynamic radii determined by FCS and DOSY experiments are in good agreement within their respective experimental errors.

Figure 6. Overlay of two separately measured DOSY spectra of AdaPEG13-b-hbPG107 (11) (red) and its mixture with 0.1 equiv of P(cyclodextrin10-co-PA7-co-HPMA58) (14) (gray).

Table 3. Diffusion Coefficients (D) and Hydrodynamic Radii (Rh) Obtained from DOSY NMR and FCS Experiments

HPMA58) and Ada-PEG13-b-hbPG107 (Figure S47c) that are not present in the NOESY spectrum of Ada-PEG13-b-hbPG107 alone (Figure S47a). Although an unambiguous assignment is hardly possible due to overlap of the β-CD and the hbPG backbone signals, these cross-correlation peaks most probably correspond to the signals of the adamantyl moiety at 1.51, 1.64, and 1.96 ppm and the inner protons H5 and H3 of β-CD, respectively. In addition, the adamantyl proton resonances experience a downfield shift upon addition of β-CD-functionalized PHPMA, typically a hint for inclusion complex formation.67 As P(cyclodextrin10-co-PA7-co-HPMA58) was labeled with a small amount of Oregon Green, the increased hydrodynamic radius of the formed supramolecular linear-g-(linear−hyperbranched) graft terpolymer can be monitored using fluorescence correlation spectroscopy (see Figure 7 and Table 3). FCS shows the β-CD grafted HPMA polymer to have a hydrodynamic radius Rh of 3.0 nm at a concentration of 3 μg/ mL (0.13 μM) in phosphate buffered saline (PBS). This value is in good agreement with the molecular weight of the polymer. Additionally, no free Oregon Green can be detected within the sample.

compound Ada-PEG13-b-hbPG107 (11) Ada-PEG13-b-hbPG107 (in the complex) P(cyclodextrin10-co-PA7co-HPMA58) (14) P(cyclodextrin10-co-PA7co-HPMA58) (in the complex)

DDOSY (m2/s)

Rh, DOSY (nm)

DFCS (m2/s)

Rh,FCS (nm)

1.8 × 10−10

1.4

9.1 × 10−11

2.7

6.9 × 10−11

3.5

7.6 × 10−11

3.0

5.1 × 10−11

4.8

5.0 × 10−11

4.9

As already proven by DOSY experiments, the concentration of the above-prepared stock solution of P(cyclodextrin10-coPA7-co-HPMA58) and Ada-PEG13-b-hbPG107 is sufficient to contain a significant amount of the supramolecular linear-g(linear−hyperbranched) graft copolymer. Knowledge of the association constant from ITC (4.8 × 104 L mol−1, Table 2) allows theoretical calculation of the associated amount of polymers and of the dilution effect on self-assembly. At a 1:11 mixture of P(cyclodextrin10-co-PA7-co-HPMA58) and AdaPEG13-b-hbPG107 and a given concentration of 16 mg mL−1 approximately 9.2 hyperbranched side chains will be associated with every polymer backbone. A dilution by a factor of 1000 reduces this value to 0.6, thus enabling disassembly of the complex by pure dilution effects. This is also reflected by results of an additional FCS experiment (see Figure S51), where the above-prepared stock solution of P(cyclodextrin10-co-PA7-coHPMA58 (0.13 mM) and Ada-PEG13-b-hbPG107 (1.5 mM) was diluted 1:1000 by pure PBS to a concentration of 0.13 μM (P(cyclodextrin10-co-PA7-co-HPMA58)) and 1.3 μM (AdaPEG13-b-hbPG107), respectively. In this case, a hydrodynamic radius similar to that of pure P(cyclodextrin10-co-PA7-coHPMA58) was detected, confirming the highly dynamic and reversible nature of the supramolecular structure.

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SUMMARY AND CONCLUSIONS We have introduced monoadamantyl-functional hyperbranched and linear−hyperbranched polyglycerols. Polymers with molecular weights ranging from 2700 to 12 100 g/mol were obtained with PDIs below 1.8. ITC analysis of the association constants of these polymers with β-CD provides insight into

Figure 7. Normalized FCS autocorrelation curves measured for Oregon Green (green triangles), P(cyclodextrin10-co-PA7-co-HPMA58) (14) (black squares), and the mixture (red circles) of P(cyclodextrin10co-PA7-co-HPMA58) (14) with an excess of Ada-PEG13-b-hbPG107 (11). The solid lines represent the respective fits with eq S2. 9551


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(3) Dodziuk, H.; Hashimoto, H.; Morillo, E.; Bilewicz, R.; Chmurski, K., Applications Other Than in the Pharmaceutical Industry. In Cyclodextrins and Their Complexes; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2006; pp 450−473. (4) Del Valle, E. M. M. Process Biochem. 2004, 39 (9), 1033−1046. (5) Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98 (5), 1875−1918. (6) Schmidt, B. V. K. J.; Hetzer, M.; Ritter, H.; Barner-Kowollik, C. Prog. Polym. Sci. 2013, http://dx.doi.org/10.1016/j.progpolymsci. 2013.09.006. (7) Davis, M. E.; Zuckerman, J. E.; Choi, C. H. J.; Seligson, D.; Tolcher, A.; Alabi, C. A.; Yen, Y.; Heidel, J. D.; Ribas, A. Nature 2010, 464 (7291), 1067−1070. (8) Zuckerman, J. E.; Choi, C. H. J.; Han, H.; Davis, M. E. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (8), 3137−3142. (9) Zeng, J.; Shi, K.; Zhang, Y.; Sun, X.; Zhang, B. Chem. Commun. 2008, 32, 3753−3755. (10) Stadermann, J.; Komber, H.; Erber, M.; Däbritz, F.; Ritter, H.; Voit, B. Macromolecules 2011, 44 (9), 3250−3259. (11) Schmidt, B. V. K. J.; Hetzer, M.; Ritter, H.; Barner-Kowollik, C. Macromolecules 2013, 46 (3), 1054−1065. (12) Hasegawa, Y.; Miyauchi, M.; Takashima, Y.; Yamaguchi, H.; Harada, A. Macromolecules 2005, 38 (9), 3724−3730. (13) Liu, H.; Zhang, Y.; Hu, J.; Li, C.; Liu, S. Macromol. Chem. Phys. 2009, 210 (24), 2125−2137. (14) Yhaya, F.; Binauld, S.; Callari, M.; Stenzel, M. H. Aust. J. Chem. 2012, 65 (8), 1095−1103. (15) Schmidt, B. V. K. J.; Hetzer, M.; Ritter, H.; Barner-Kowollik, C. Polym. Chem. 2012, 3, 3064−3067. (16) Huan, X.; Wang, D.; Dong, R.; Tu, C.; Zhu, B.; Yan, D.; Zhu, X. Macromolecules 2012, 45 (15), 5941−5947. (17) Zhang, Z.-X.; Liu, X.; Xu, F. J.; Loh, X. J.; Kang, E.-T.; Neoh, K.G.; Li, J. Macromolecules 2008, 41 (16), 5967−5970. (18) Tao, W.; Liu, Y.; Jiang, B.; Yu, S.; Huang, W.; Zhou, Y.; Yan, D. J. Am. Chem. Soc. 2012, 134 (2), 762−764. (19) Bertrand, A.; Stenzel, M.; Fleury, E.; Bernard, J. Polym. Chem. 2012, 3 (2), 377−383. (20) Yan, J.; Li, W.; Liu, K.; Wu, D.; Chen, F.; Wu, P.; Zhang, A. Chem.Asian J. 2011, 6 (12), 3260−3269. (21) Gao, C.; Yan, D. Prog. Polym. Sci. 2004, 29 (3), 183−275. (22) Voit, B. I.; Lederer, A. Chem. Rev. 2009, 109 (11), 5924−5973. (23) Wilms, D.; Stiriba, S.-E.; Frey, H. Acc. Chem. Res. 2010, 43 (1), 129−141. (24) Kainthan, R. K.; Janzen, J.; Levin, E.; Devine, D. V.; Brooks, D. E. Biomacromolecules 2006, 7 (3), 703−709. (25) Imran ul-haq, M.; Lai, B. F. L.; Chapanian, R.; Kizhakkedathu, J. N. Biomaterials 2012, 33 (35), 9135−9147. (26) Calderón, M.; Quadir, M. A.; Sharma, S. K.; Haag, R. Adv. Mater. 2010, 22, 190−218. (27) Schömer, M.; Schüll, C.; Frey, H. J. Polym. Sci., Part A: Polym. Chem. 2013, 51 (5), 995−1019. (28) Wurm, F.; Frey, H. Prog. Polym. Sci. 2011, 36 (1), 1−52. (29) Nuhn, L.; Schüll, C.; Frey, H.; Zentel, R. Macromolecules 2013, 46 (8), 2892−2904. (30) Lach, C.; Hanselmann, R.; Frey, H.; Mülhaupt, R. Macromol. Rapid Commun. 1998, 19 (9), 461−465. (31) Lam, D.; Little, S.; Rutherford, J.; Twyman, L. J.; Zheng, X. Macromolecules 2008, 41 (5), 1584−1586. (32) Kuo, P.-L.; Ghosh, S. K.; Liang, W.-J.; Hsieh, Y.-T. J. Polym. Sci., Part A: Polym. Chem. 2001, 39 (17), 3018−3023. (33) Pargen, S.; Omeis, J.; Jaunky, G.; Keul, H.; Möller, M. Macromol. Chem. Phys. 2011, 212 (16), 1791−1801. (34) Schüll, C.; Frey, H. ACS Macro Lett. 2012, 1 (4), 461−464. (35) Schüll, C.; Nuhn, L.; Mangold, C.; Christ, E.; Zentel, R.; Frey, H. Macromolecules 2012, 45 (15), 5901−5910. (36) Desai, A.; Atkinson, N.; Rivera, F.; Devonport, W.; Rees, I.; Branz, S. E.; Hawker, C. J. J. Polym. Sci., Part A: Polym. Chem. 2000, 38 (6), 1033−1044. (37) Helms, B.; Mynar, J. L.; Hawker, C. J.; Fréchet, J. M. J. J. Am. Chem. Soc. 2004, 126 (46), 15020−15021.

the shielding effect of the hyperbranched moiety. As a key result of this work, the association constant was found to drop with increasing molecular weight of the hyperbranched polyglycerol, but levels to a final value already at moderate molecular weights of the hyperbranched block. Additionally, the presence of the PEG spacer between the adamantyl group and the hyperbranched polyglycerol resulted in significantly increased association constants, demonstrating the steric effect of branching. Cyclodextrin-functionalized PHPMA was synthesized using RAFT techniques, subsequent reactive ester, and click chemistry. The postpolymerization modifications also enable fluorescent labeling with Oregon Green for diffusion/size characterization. Mixing of this water-soluble HPMA polymer with pendant cyclodextrin units and the monoadamantylfunctionalized poly(ethylene glycol)-block-hyperbranched polyglycerols yields a supramolecularly assembled linear-g-(linear− hyperbranched) graft terpolymer. The structure represents the first self-assembled linear-graf t-hyperbranched polymer structure based on adamantane and cyclodextrin and was proven by ITC, DOSY, and FCS experiments. This modular approach provides building blocks that can be used for a wide range of architectures. PHPMA39,43,44 and hyperbranched poly(glycerol)24 are highly biocompatible, while cyclodextrins find manifold applications as pharmaceutical solubilizers.1 First cyclodextrin−adamantane-based polymeric nanostructures have recently been successfully applied in clinical tests as advanced siRNA drug delivery carrier systems.7 Taking these aspects into account, the reversible supramolecular structure of the presented graft terpolymer allows its disassembly into smaller subunits, which may show improved kidney clearance and thus may make the presented concept attractive for potential biomedical applications as a drug carrier.

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ASSOCIATED CONTENT

S Supporting Information *

Additional instrumentation, experimental procedures, NMR and UV/vis spectra, SEC elugrams, MALDI-TOF spectra, and ITC data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.M. thanks the Graduate School of Excellence Materials Science in Mainz “MAINZ” (DFG/GSC 266) for financial support and thanks Ulrike Kemmer-Jonas, Markus Paulen, and Meike Schinnerer for technical assistance. L.N. and A.T. are grateful to the Fonds der Chemischen Industrie and the Max Planck Graduate Center with the Johannes GutenbergUniversität Mainz (MPGC) for financial funding.

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REFERENCES

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