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Multifunctional and Dual-Responsive Polymersomes as Robust Nanocontainers: Design, Formation by Sequential PostConjugations, and pH-Controlled Drug Release Banu Iyisan,†,‡,§ Jörg Kluge,†,‡ Petr Formanek,† Brigitte Voit,*,†,‡,§ and Dietmar Appelhans*,† †

Leibniz Institute for Polymer Research Dresden, Hohe Strasse 6, D-01069 Dresden, Germany Organic Chemistry of Polymers, Technical University of Dresden, D-01062 Dresden, Germany § International Helmholtz Research School for Nanoelectronic Networks, 01328 Dresden-Rossendorf, Germany ‡

S Supporting Information *

ABSTRACT: Robust, multiresponsive, and multifunctional nanovesicles are in high demand not only as carrier systems but also for applications in microsystem devices and nanotechnology. Hence, multifunctional, pH-responsive, and photo-cross-linked polymersomes decorated with adamantane and azide groups are prepared by mixed selfassembly of suitably end-modified block copolymers and are used for the subsequent postconjugation of the polymersome surface by using covalent and noncovalent approaches. For the covalent approach, nitroveratryloxycarbonyl-protected amine (NVOC) molecules as lightresponsive moieties are introduced into the polymersomes through an azide−alkyne click reaction. After photocleavage of NVOC units, functional dye molecules react with the now freely accessible amine groups. The noncovalent approach is performed subsequently to introduce further moieties, making use of the strong adamantane-β-cyclodextrin host−guest interactions. It is quantitatively proven that all reactive groups have sufficient accessibility as well selective and orthogonal reactivity throughout these stepwise processes to allow the successful establishment of aimed pH- and light-responsive multifunctional polymersomes. Moreover, this sequential methodology is also applied to obtain doxorubicin-loaded multifunctional polymersomes for an efficient pH-controlled drug release. Overall, tunable membrane permeability combined with the potential for introducing multiple targeting groups by light-exposure or host−guest interactions make these smart polymersomes promising nanocontainers for many applications.

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INTRODUCTION Over the past few decades, interdisciplinary nanotechnology research has played an essential role in understanding the behavior of complex natural biomacromolecules by creating their synthetic analogues. One of the noteworthy attempts at this is the development of lipid-based vesicles, so-called liposomes, that mimic the cell membrane.1 Although these nanoparticles have been very attractive in many biotechnological applications, they lack stability and allow for limited chemical functionalization.2 Therefore, inspired by them, their polymeric analogues, named polymersomes, have been invented within the last couple of decades.3 Polymersomes are comprised of a bilayer membrane like their lipid counterparts.4 However, their thicker membrane made with the aid of synthetic amphiphilic block copolymers leads to a more stable and robust system.5 Because of these outstanding advantages, nowadays they draw wide interest in various fields of biomedical applications, including drug delivery,6−8 synthetic biology,9−11 and biosensing.12,13 Nevertheless, to be effective in these mentioned areas, polymersomes must have some key features, such as higher mechanical strength, a stimuli responsive membrane, and © XXXX American Chemical Society

selective targeting moieties on their surface. Although some of those properties have already been introduced individually in many polymersomes, it is rare to combine all of these abilities into one single system. For instance, longevity in the cardiovascular system of a human body is a significant issue that has to be supplied for many biomedical purposes.14 Selfassembled polymersomes can encounter disassembly problems in blood circulation due to various physiological factors, including shear forces or dilution effects.15,16 For this problem to be overcome, an efficient way is cross-linking of the bilayer membrane by integrating corresponding units to the block copolymer structure.17 Recent improvements to achieve photocross-linked vesicles have been performed by using 3,4dimethyl maleic imidoethyl methacrylate11,18 and 2-hydroxy4-(methacryloyloxy) benzophenone (BMA) units within a short time of UV irradiation.19 Another essential issue especially in the field of drug delivery is to establish tunable membrane permeability. For enhancing Received: December 29, 2015 Revised: February 5, 2016

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DOI: 10.1021/acs.chemmater.5b05016 Chem. Mater. XXXX, XXX, XXX−XXX

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Chemistry of Materials

Figure 1. Schematic overview of the sequential postconjugation processes on a polymersome surface using a covalent and noncovalent approach in combination with chemical structures of block copolymers having azide (BC1), methoxy (BC2), and adamantane (BC3) functionalities. Covalent approach: conversion of PS1 into PS1C, PS1C into PS1D, and PS1D into PS1R. Noncovalent approach: conversion of PS1R into PS1H.

the therapeutic efficiency and avoiding toxic effects, it is important to release the drug at only targeted regions of the body. This can be realized simply by incorporating stimuli responsive segments to the polymersome structure. Several studies to prepare such systems responding to internal or external stimuli like pH,20 temperature,21 redox,22 magnetic field,23 and light24 have been reported. Although such triggers provide controlled release of the drug by changing permeability or morphology of the membrane, they are still inadequate for specific targeting to certain disease sites.14 Therefore, active targeting via a specific receptor−ligand interaction can be a promising approach to obtain enhanced therapeutic efficiency

as well as a diagnostic ability. This approach can be achieved by introducing targeting ligands to the surface of the polymersome membrane.25,26 In this respect, attachment of many ligands, such as antibodies,27−29 peptides, 30 folic acid, 6,31 and sugars,32,33 have been studied. Conjugation of these ligands can be performed by functionalization of the polymersome surface with reactive groups. Several approaches for polymersome functionalization have been reported, including biotin− streptavidin binding,34 adamantane−cyclodextrin binding,35,36 azide−alkyne click chemistry,32,37−39 and conjugation via a bisaryl hydrazine bond.13 Although these studies have resulted in feasible functionalized polymersomes, further efforts are B


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cyanine 7 NHS ester was provided from Lumiprobe GmbH (Germany). Dialysis membranes made of regenerated cellulose (molecular weight cutoffs of 5000 and 50000) were purchased from Carl Roth. 1-Aminoadamantane, 4,5-dimethoxy-2-nitrobenzyl chloroformate, propargylamine, sodium ascorbate, copper(II)sulfate pentahydrate, copper(I) bromide, copper(I) iodide, 2-bromoisobutyryl bromide, 2,2′-bipyridine, aluminum oxide (neutral, activated), poly(ethylene glycol) methyl ether (PEG-OH; Mn: 2000 g/mol; Đ: 1.05), 2-(diethylamino)ethyl methacrylate (DEAEMA), rhodamine B isothiocyanate, tris(benzyltriazolylmethyl) amine (TBTA), 2-butanone (Fluka), triethylamine, N,N-dimethylformamide, N,N-diisopropylethylamine (DIPEA), and dimethyl sulfoxide were purchased from Sigma-Aldrich. Doxorubicin hydrochloride (Dox) was acquired from Carbosynth Limited. All other chemicals were obtained from Acros Organics, and anhydrous solvents were stored over molecular sieves. Dynamic Light Scattering (DLS). DLS measurements were performed at 25 °C using a Zetasizer Nanoseries instrument (Malvern Instruments, UK) equipped with a multipurpose autotitrator and a 633 nm He−Ne laser at a fixed scattering angle of 173°. The size of the polymersomes is given as intensity-average diameter (zaverage) values assuming the refractive index of the polymer is 1.50. Zeta potential of the polymersomes was determined by titration of polymersomes against HCl (0.1 mol/L) and KOH (0.1 mol/L) solutions. During this titration, the size of the polymersomes was also recorded to observe the stability and swelling behavior of the vesicles. Cryogenic Transmission Electron Microscopy (cryo-TEM). Cryo-TEM images were obtained using a Libra 120 microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) at an acceleration voltage of 120 kV. Samples were prepared by dropping 1 μL of polymersome solution (1 mg/mL) on each side of a copper grid coated with holey carbon foil (so-called Lacey type). A piece of filter paper was used to remove the excess water, and the sample was then rapidly frozen in liquid ethane at −178 °C. The blotting with the filter paper and plunging into liquid ethane was done in a Leica GP device (Leica Microsystems GmbH, Wetzlar, Germany). All images were recorded in bright field at −172 °C. Formation of Polymersomes. A mixture of block copolymers was dissolved in aqueous HCl solution (pH 2) and then passed through a 0.2 μm nylon filter to remove any impurities. To initiate the self-assembly process, the pH was slowly increased to pH 9 by adding 1 M NaOH. Finally, polymersomes were formed after 4 days of stirring in dark conditions. Cross-Linking of Polymersomes. The polymersome solution was passed through the 0.8 μm nylon filter and was then placed in the UV chamber (UVACUBE 100, honle UV Technologies, Germany) and irradiated for 30 min. NVOC Modification of Polymersomes. Previously published methods were modified as follows:37,42 The aqueous solutions of CuSO4·5H2O (0.25 mol eq), sodium ascorbate (0.5 mol eq), TBTA (0.25 mol eq in DMSO), and alkyne-modified NVOC groups (1.5 mol eq in DMSO, see Supporting Information) were added to the 1 mg/ mL polymersome solution at pH 8 (PS1 or PS2, azide groups, 1 mol eq). The reaction mixture was stirred for 2 days at room temperature. Subsequently, the polymersome solution was transferred to a dialysis membrane (5 kDa MWCO) and extensively dialyzed against EDTA solution in Millipore water (0.055 mM, pH 8) for 2 days. Photocleavage of NVOC Groups via UV Irradiation. The polymersome solutions were irradiated at a distance of 1 cm from the UV lamp in the range of 0−600 s (Omnicure S2000, Lumen Dynamics Group Inc., Canada). The decrease in absorbance at 345 nm was monitored by UV−vis spectroscopy. Then, the final polymersome solution was dialyzed against water (pH 8) for 4 h to remove byproducts of the deprotection reaction. Determination of NVOC Functionalization Degree. A similar method as described previously was applied as follows:37 PS2C polymersome was freeze-dried and dissolved in CHCl3 at different concentrations. Then, the absorbances of the prepared solutions were measured at 345 nm using UV−vis spectroscopy and fitted to a linear line (A = 6289.52c, R2 = 0.997, Figure S16b). For the comparative relationship to be obtained, the same approach was also applied to a

necessary to combine various reactive groups into one single polymersome system. To date, most reported examples for postconjugation of polymersomes are focused on a single conjugation reaction by using noncovalent or covalent binding methods.25,26 Only a few studies report on two conjugation steps,27−29 e.g., biotinylated sialyl-Lewis X or biotinylated antiICAM-1, or both simultaneously, were applied for the formation of leukocyte-mimicking polymeric vesicles via avidin−biotin conjugation.28,29 The design of multi- and heterofunctional vesicles in combination with the above-mentioned key properties will provide robust systems with simultaneous recognition and delivery ability for intelligent nanocarriers and nanocontainers. Moreover, the establishment of polymersomes for selective and sequential surface modification will also increase their use, for example, in the field of diagnostics, synthetic biology, lab-on-achip, and microsystem applications where selective functionalization of polymersomes on the outer membrane surface but also inside the cavity is of high interest. Thus, this motivated us to develop multifunctional photo-cross-linked polymersomes with pH and light responsivity as well as various reactive groups on their surface. As illustrated in Figure 1, we performed two primary functionalization strategies making use of covalent and noncovalent bindings. This includes first the use of a mixture of two differently end group-functionalized block copolymers followed up by a coassembly process with nonfunctionalized block copolymer and cross-linking after polymersome formation. The further 4-fold conjugation process was started with azide−alkyne click chemistry to initiate the covalent approach and finalized by the noncovalent approach based on host−guest interactions. Therefore, the surface of polymersomes was designed to have azide and adamantane groups for supporting these conjugation steps. The covalent conjugation approach was used to integrate nitroveratryloxycarbonyl (NVOC)protected amine groups (Figure 1) to add light responsiveness for controlled surface functionalization. NVOC derivatives are one of the most promising photolabile protection groups for amino functionalities with a cleavage ability via UV exposure.40,41 Thus, a photocleavage reaction on a polymersome surface resulted in amine functionalities followed by linking a dye via an isothiocyanate-amine reaction. In addition, this sequential approach was also extended to drug encapsulation and subsequent in vitro release at endosomal (pH 5) and extracellular (pH 7.4) physiological conditions. This release study was performed utilizing doxorubicin as a drug molecule on NVOC-conjugated polymersomes before (PS1C-Dox) and after (PS1D-Dox) the photocleavage process to verify the concept of dual responsivity from both a light and pH perspective. Thus, the aimed robust, multifunctional, and dual responsive polymersomes were established by demonstrating the accessibility of various functional groups as well as their selective and orthogonal reactivity throughout the whole sequential reaction steps combined with pH-dependent drug release.

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EXPERIMENTAL SECTION

Materials. Block copolymers were synthesized by ATRP and detailed procedures are given in the Supporting Information. Azideterminated poly(ethylene glycol) (N3PEG-OH; Mn: 2700 g/mol; Đ: 1.18) was purchased from Polymer Source (Canada). 2-Hydroxy-4(methacryloyloxy) benzophenone (BMA) was obtained from Alfa Aesar. 6-Monodeoxy-6-monoamino-β-cyclodextrin was received from Cyclodextrin Shop (Division of AraChem, Netherlands). SulfoC


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Chemistry of Materials model compound (NVOC-PEG60OH, M1C) that had 50% NVOC functionalization (XM1C) determined from the 1H NMR spectrum (A = 3330.33c, R2 = 0.998, Figure S16a). Subsequently, the ratio of the slopes of the regression lines was related with the functionalization degree of the two compounds (1.88XPS2C = XM1C) to estimate the relative amount of NVOC modification on the polymersome surfaces. Determination of Photocleavage Efficiency. The PS2C polymersome was irradiated (600 s) and dialyzed (against pH 8 for 4 h) as indicated previously. The polymersome solutions before and after irradiation were freeze-dried and dissolved in acidic water (concentration was 1 mg/mL) for UV−vis characterization. The absorbance values at 345 nm were obtained, and the photocleavage efficiency was determined by calculating the amount of NVOC chromophore cleavage (Figure S17 and eq 1). Rhodamine B NCS Modification of Polymersomes. To a 1 mg/mL polymersome solution at pH 8 (PS1D) was added 19 μL of Rhodamine B NCS solution in DMSO (RhB-NCS, 0.03 mM). The reaction mixture was stirred for 12 h at room temperature, and then the polymersome solution was extensively dialyzed against water (pH 8) for 3 days using a 50 kDa MWCO membrane. The same process was also applied to the PS1 polymersomes as a negative control. The concentration of NH2 groups reacted with RhB-NCS dye was determined by using the subtracted absorbance value of PS1R and PS1 polymersomes at 555 nm and the measured molar absorption coefficient (εRhNCS = 136811 at 555 nm) of RhB-NCS in polymersome solutions (Figure S18a). Subsequently, the reactivity of NH2 groups on the polymersome surface was determined by eq 2. β-Cyclodextrin Modification of Polymersomes. Aqueous solution of sulfo-cyanine7 labeled cyclodextrin (β-CD-Cy7, 6 mol eq) was added to polymersomes that were already reacted with Rhodamine B NCS fluorescent dye (PS1R, Ada groups 1 mol eq). After 20 h of stirring at room temperature, the polymersome solution was transferred to a dialysis membrane (MWCO: 50 kDa) and dialyzed extensively against water (pH 8) for 3 days. As a control experiment, the same procedure was also applied to polymersomes without any adamantane groups (PS0) and also PS1 polymersomes with adamantane groups. The conjugation efficiency of β-CDCy7 molecules was determined with a similar strategy as done for the evaluation of amino groups. The molar absorption coefficient of βCDCy7 was measured as εCDCy7 = 113964.8 at 775 nm in polymersome solutions (Figure S18b) and used to calculate the accessible adamantane groups on the PS1H surface from eq 3. Preparation of Doxorubicin-Encapsulated Polymersomes. Doxorubicin encapsulation was performed during the polymersome formation process with a preloading approach. A mixture of block copolymers was dissolved in aqueous HCl solution (pH 2) and then passed through a 0.2 μm nylon filter as described previously. Afterwards, the pH was increased to a value of pH 5 by utilizing 1 M NaOH solution. Doxorubicin (3 mg, 0.5 mL in Millipore H2O) was added to the block copolymer solution in this step followed by increasing the pH to 7.4 to initiate the self-assembly process. The final concentration of the polymer was adjusted to 1 mg/mL, whereas the Dox concentration was 0.13 mg/mL. After stirring the mixture for 4 days in the dark, polymersomes were photo-cross-linked for 30 min and then purified using a hollow fiber filtration (HFF, SpectrumLabs, USA) system to remove the nonencapsulated Dox molecules. The purification was performed using modified poly(ether sulfone) (mPES)-based filter modules (MWCO: 100 kDa) with a transmembrane pressure (TMP) of 130 mbar by washing the samples with phosphate buffer at pH 7.4 for several cycles. To calculate the encapsulation efficiency, all wash solutions during purification were collected, and the absorbance at λ = 480 nm was measured with UV− vis spectroscopy. From the calibration curve at pH 7.4 (Figure S21a), the amount of free Dox was defined, and the encapsulation efficiency (71.9 ± 0.4%) was determined by means of eq 4. To obtain NVOC-modified Dox-loaded polymersomes (PS1CDox), the same protocol was applied to PS1-Dox polymersomes like that for PS1 vesicles performed previously. Here, only the reaction medium was retained at pH 7.4 rather than pH 8 to avoid any Dox instability. As the final step, some portion of PS1C-Dox polymersomes

were used in photocleavage process to obtain PS1D-Dox vesicles for further release studies. In Vitro Doxorubicin Release. Dox-encapsulated polymersomes at pH 5 and 7.4 (4 mL, PS1C-Dox and PS1D-Dox) were poured into dialysis tubes (MWCO 5000), which were then placed in 2 L buffercontaining beaker at constant temperature (37 °C) and stirring (200 rpm) conditions. Two different release media were prepared as acetate buffer (0.01 M) for pH 5 profile and phosphate buffer (0.01 M) for pH 7.4 profile. For determining the drug release, 0.8 mL of sample was taken at selected time intervals for UV−vis analysis (λ = 480 nm) and returned back to the dialysis membrane after the measurement. The amount of remaining Dox at each sampling point was then calculated from the calibration curves at pH 5 and 7.4 (Figure S21). Finally, the cumulative Dox release at each time interval was obtained from eq 5.

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RESULTS AND DISCUSSION

Preparation and Characterization of Polymersomes. One common method to prepare multifunctional polymersomes is to conjugate desired functional moieties to the block copolymer structure before starting the self-assembly process for polymersome formation. Thus, three block copolymers (Figure 1) having azide, adamantane, and methoxy end groups at their hydrophilic segment were synthesized using atom transfer radical polymerization (ATRP) (Figure S1). The structures of the block copolymers are based on poly(ethylene glycol)-b-poly[2-(diethylamino)ethyl methacrylate-stat-2-hydroxy-4-(methacryloyloxy)benzophenone] (BC2), which was previously studied by our group.6,19 Poly(ethylene glycol) (PEG) is the hydrophilic segment of the amphiphilic block copolymer, whereas pH sensitive 2-(diethylamino)ethyl methacrylate (DEAEM) and photo-cross-linker benzophenone (BMA) groups together form the hydrophobic part. As shown in Figure 1, the hydrophobic block length of the polymers was kept almost equal to induce homogeneous formation for the hydrophobic part of the polymersome membrane. However, the hydrophilic length of the azide- and adamantane-functionalized block copolymers (BC1, BC3) was designed to be longer than the nonfunctional methoxyterminated block copolymer (BC2) to increase the accessibility of the reactive groups on the polymersome surface. This strategy has recently been successfully applied in our group to conjugate folate groups to the polymersome system, which showed remarkable specificity toward cancer cells.6 Thus, polymersomes were formed by using the pH switch method in which the mixture of the polymers were selfassembled in aqueous media without the aid of any organic solvent.43 In brief, tertiary amine groups of the PDEAEM segment are protonated in acidic conditions, which enables them to dissolve the mixture of BC1 (29 mol %), BC2 (61.8 mol %), and BC3 (9.2 mol %) block copolymers in acidic water (pH 2). Then, to induce the self-assembly process, deprotonation of the tertiary amine moieties is performed by simply increasing the pH to a basic state (pH 9). After 4 days of stirring, the final polymersome structure is formed with a bilayer membrane having a central hydrophobic part based on PDEAEM and BMA segments and a hydrophilic inner and outer PEG corona with azide and adamantane functionalities. Our group previously investigated different photo-cross-linkers and their positive effects on preserving the shape of the vesicles against different stimuli.11,19 For applying several reaction sequences to a single polymersome system (Figure 1), robust and mechanically stable polymersomes are needed. Therefore, BMA moieties were used for cross-linking the membrane of the polymersomes by UV irradiation with a low intensity UV lamp D


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Figure 2. (a) Reversible swelling−shrinking of polymersomes (PS1) upon changes in pH value. (b) pH-dependent zeta potential and diameter variation of polymersomes (PS1). (c, e) Cryo-TEM micrographs of polymersomes (PS1) at pH 9 and pH 5. (d) Membrane thickness distribution of polymersomes (PS1) determined from corresponding cryo-TEM micrographs.

Table 1. Specification of the Polymersomes with Different Functional Groups functional groups [mol %] polymersome

N3

PS0 PS1 PS1C PS1D PS2 PS2C PS2D

29.0 21.3 21.3 100 73.5 73.5

NVOCa

NH2a

Ada

7.7 3.2

4.5

9.2 9.2 9.2

26.5 11

15.5

photo- cross-linked yes yes yes yes no no no

diameterb [nm] 117 120 117 119 88.0 84.5 93.5

± ± ± ± ± ± ±

1.1 1.6 0.2 0.3 0.2 0.5 0.1

PDIc 0.19 0.20 0.20 0.20 0.15 0.18 0.14

± ± ± ± ± ± ±

0.03 0.03 0.01 0.01 0.02 0.02 0.02

a

These values represent the functionalities only at the outer surface of the membrane. bDiameter is measured by DLS at pH 8. cPDI = polydispersity index of polymersomes that shows the size variation.

the interior and outer part of the bilayer membrane during the formation of surface-functionalized polymersomes when using at least two different block copolymers. Having sufficient functional groups on the outer region of the polymersomes is a well-known requirement for starting a postconjugation process on the polymersome surface. Otherwise, no successful administration of polymersomes in drug delivery with selective targeting can be achieved.25−31 As a next step, the stability of polymersomes was investigated at various pH conditions. The size and zeta potential values from pH 10.1 to 3.3 were obtained using DLS equipped with an autotitrator (Figure 2b). DLS results show that polymersomes preserve their shape without disassembly at acidic conditions because of the cross-linked membrane. In addition, the expected size change of the polymersomes at approximately pH 7 roughly corresponds to the pKa of the DEAEM groups.44 This result was also supported by zeta potential measurements, revealing that the membrane is almost unprotonated at physiological pH conditions. Furthermore, the cross-linking process also provides tunable membrane permeability to the multifunctional polymersome system. This was explored by

for 30 min. The polymeric vesicles were characterized by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM) analysis (Figure 2). The size distributions as well as the ratios of the functional moieties of the formed polymersomes are presented in Table 1. From Table 1, DLS results also show that integrating functional moieties on the polymersome surface by the coassembly process of differently end-functionalized block copolymers has no significant effect on the average diameter of the vesicles. Additionally, low PDI values in the range of 0.14 to 0.20 demonstrate the low dispersity of the vesicles even when 9.2 mol % of adamantane-terminated polymers and 29 mol % of azide-terminated polymers were used for the formation of polymersomes. This impressively implies two key issues for the polymersome formation: First, the chosen block copolymer ratio does not induce any undesired side effects like individual self-assembly or aggregation problems. However, purposefully, only a maximum of 9.2 mol % of BC3 was incorporated due to the hydrophobic nature of adamantane groups. Second, it can be assumed, as indicated in previous studies,6,28,29,34 that a homogeneous distribution of functional groups takes place in E


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Figure 3. (a) Reaction scheme of the NVOC conjugation and cleavage process on PS1 polymersomes. (b) UV−vis spectra of PS1C polymersomes upon UV exposure up to 600 s showing the decrease of absorption bands due to NVOC cleavage. (c) The absorbance change (%) of PS1 and PS1C at λ = 345 nm versus irradiation time under identical UV exposure. (d) Intensity size distribution of cross-linked polymersomes PS1, PS1C, and PS1D.

following the size change of the polymersomes upon changes in pH value from basic to acidic for 5 cycles (Figure 2a). The multifunctional polymersome was able to swell and shrink reversibly at acidic and basic conditions because of the protonation/deprotonation of the PDEAEM groups in the membrane. Here, the diameter of the vesicles increases approximately 44.2% in the swollen state. Similar pHdependent reversible swelling behavior for photo-cross-linked polymersomes consisting of two different block copolymers was found previously and provided the desired permeability during the drug delivery process in cancer cells.6 Cryo-TEM investigation was conducted to confirm the vesicular structure of the polymersomes at both acidic and basic conditions (Figure 2c−e). Polymersome visualization by cryoTEM has a great advantage over conventional TEM methods because the native vesicle structure can be imaged in the frozen hydrated state without the aid of sample staining. Sufficient contrast between the polymeric membrane and the vesicle interior filled with water leads to a clear identification of the structure. This gave us the opportunity to determine the membrane thickness of polymersomes at high and low pH values by analyzing several cryo-TEM micrographs (Figure 2d). The average membrane thickness increases from 18.6 ± 1.7 to 26.8 ± 3.8 nm when changing pH from a basic state (pH 9) to an acidic value (pH 5). This impressively implies that the repelling of polymer chains by protonation of DEAEM segments at acidic conditions causes the state of a permeable membrane in polymersome PS1 with azide and adamantane

surface groups (Figure 1 and Figure 2d). The increase of the membrane thickness was calculated to be 44.1%. This is in a perfect agreement with our previous swelling/shrinking experiments as stated above (Figure 2a). Thus, the cryo-TEM study allowed us to consider the permeable polymersome membrane like a gate that can be opened or closed by the trigger of pH stimulus. Additionally, it was proven that the average thickness of the polymersome membrane was almost 3.5 times thicker than the phospholipid membrane of liposomes, which in turn leads to more stable, tougher, and in the collapsed state, tighter polymeric vesicles.45−47 Covalent Conjugation Approach (PS1 to PS1D): Surface Functionalization of Polymersomes with Photoactive Moieties and the Successive Photocleavage Process. A common way to functionalize a polymersome surface is the introduction of functional moieties to the preformed vesicles after the block copolymer assembly. Herein, we applied this technique for further postfunctionalization of pH-sensitive polymersomes with photoactive moieties to obtain a multiresponsive structure (Figure 1: from PS1 to PS1R). The aim was to introduce amino groups to the polymersome surface that are protected by the well-known photolabile NVOC group for selective cleavage by UV irradiation at longer wavelengths up to 410 nm.48 The strategy was to selectively obtain free amine groups on the vesicle surface only after a light trigger, which offers the potential to selectively bind biological entities afterwards. Therefore, the postmodification process of the photo-cross-linked polymersomes PS1 was started with the F


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Figure 4. (a) Reaction scheme of NVOC conjugation on PS2 polymersomes via identical click modification as done for PS1 polymersomes. (b) UV−vis spectra of freeze-dried PS2 and PS2C in CHCl3.

Thus, this clearly implies that BMA units show overlapping absorption bands with NVOC groups in the region of 250 and 330 nm due to their π−π* transition and n−π* transition, respectively.51 In addition, photoreduction of the carbonyl groups in BMA units upon UV exposure could also lead to a decrease in the absorption spectra.51,52 To distinguish the effects of these two photoactive species in the same absorption wavelengths range, the same irradiation process to polymersomes without NVOC groups (PS1) was applied. As expected, the absorbance decrease (λ = 345 nm) of NVOC-conjugated polymersomes (PS1C) is approximately 1.5 times higher than that of those without NVOC groups (PS1) (Figure 3c). The small deviation of three repeated irradiation processes from different batches of PS1 and PS1C polymersomes supports the reproducible nature of the photocleavage reaction as well as the NVOC conjugation (Figure 1, Figure 3c). Furthermore, the shape persistence and stability of polymersomes during this whole reaction sequence was investigated by DLS and cryoTEM visualization (Figure 3d, Table 1, and Figure 5c, d). This is an important prerequisite to use those polymeric vesicles as stable nanocontainers for various applications. Results from the DLS study demonstrate that the average diameter as well as the monodisperse nature of the polymersomes is not altered during these reaction processes. Additionally, cryo-TEM images of PS1D polymersomes at basic (pH 8) and acidic (pH 5) states confirm this claim by showing the preserved vesicular structure of the polymersomes, as seen in Figure 5c, d. For further proof of this conjugation step (Figure 1: from PS1 to PS1C), two model reactions under identical conditions were performed (Figure 4a, Figure S15). First, azide-terminated polymersomes without BMA units (PS2) were prepared by the self-assembly of the corresponding block copolymer (BC4). Following this, the same click conditions were conducted to obtain NVOC-conjugated polymersomes (PS2C). Considering the UV−vis spectra of polymersomes PS2 and PS2C (Figure 4b), absorption bands in the region of λ = 260−400 nm after the click reaction are clear evidence of NVOC conjugation on the surface of PS2. In addition, the characterization of the freeze-dried and disassembled PS2C polymersomes by 1H NMR spectroscopy supports the successful conjugation of NVOC on the PS2 surface by attributed 1H NMR signals

synthesis of NVOC-protected amine molecules possessing an alkyne moiety (see SI). Afterwards, this molecule was conjugated to the azide groups on the polymersome surface of PS1 in the presence of a copper catalyst and tris(benzyltriazolylmethyl) amine(TBTA) as a ligand using slightly modified literature procedures.37,42 After 2 days of reaction time, unbound NVOC groups and the copper catalyst were removed by extensive dialysis. For guaranteeing that the purification was efficient, the same amount of NVOC molecules without polymersomes was dialyzed under identical conditions, and there was no sign of NVOC absorption in the UV−vis spectra (see SI). After verification of the successful purification, the resulting polymersomes (PS1C) were exposed to UV light (320−500 nm) for up to 600 s. The chemistry of this reaction sequence (PS1 over PS1C to PS1D) is outlined in Figure 3a. Thus, the photocleavage of the NVOC chromophore in PS1C resulted in the formation of free amine moieties on the polymersome surface of PS1D. Although the chemistry is straightforward, confirmation of this conjugation step as well as the cleavage process required multiple characterization efforts and model studies. Thus, the first inspection was realized by the time-dependent UV−vis study of the NVOC-conjugated polymersomes (PS1C) (Figure 3b). The characteristic chromophore absorption of NVOC groups is seen in the wavelength range between 260 and 400 nm, where the maximum absorption is around λmax =345 nm.49,50 As illustrated in Figure 3b, UV−vis spectra recorded during the irradiation process show a sharp decrease of the corresponding absorption bands. Although this is strong evidence for the cleavage of the NVOC chromophore and its conjugation to the vesicle, the effects of photo-cross-linker (BMA) units in the polymersome membrane has to be considered due to similar absorption maxima of BMA compared to NVOC. The UV−vis spectra of PS1C (Figure 3b) clearly indicates that there was no significant absorbance change after 500 s, and the absorption bands between 300 and 400 nm did not fully disappear. This remaining absorbance can be caused by the residual photodegration products, such as nitrosobenzaldehyde, to some extent.49 However, the significant contribution was originated by BMA units because of the higher molar ratios compared with the possible side products. G


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Figure 5. (a) Reaction scheme of RhB-NCS conjugation on PS1D polymersomes. (b) Monitoring of RhB-NCS coupling to polymersomes through amino groups (PS1R) with UV−vis analysis during the dialysis procedure. (c) UV−vis spectra of RhB-NCS-containing polymersomes with (PS1R) and without (PS1) NH2 groups to show the covalent conjugation of the dye. (d, e) Cryo-TEM micrographs of PS1D polymersomes at pH 8 and 5. (f, g) Cryo-TEM micrographs of PS1R polymersomes at pH 8.

functionalities at both the interior and exterior of the polymersomes. As a next step, the photocleavage efficiency was determined as 58.4 ± 2.3% by calculating the NVOC chromophore cleavage from UV−vis analysis using PS2C polymersomes (Figure S17). This incomplete conversion behavior might be explained by imine formation resulting from a reaction of nitroso benzaldehyde with amine groups released on the polymersome surface. For this condition to be avoided, the polymersomes were immediately dialyzed after UV exposure. However, dialysis required a certain amount of time. Thus, this side reaction could occur before all aldehyde products were removed. The outcome is also in agreement with previously published results where del Campo et al. obtained 40−50% deprotection efficiency even in the presence of a carbonyl scavenger.48 From a theoretical point of view, when transferring results from PS2D to PS1D, the final content of various functional groups in polymersomes is presented in Table 1. Thus, PS1D may have approximately 4.5 mol % of free NH2 groups on the surface as an additional functionality for the next conversion step (Figure 1: PS1D to PS1R). Covalent Approach on the Polymersome Surface: Conjugation of the Dye as the Final Step (PS1D to PS1R). Gathering information regarding the accessibility of converted functional groups at each conjugation step on the polymersome surface is of great significance to use them as efficient nanocontainers for various applications. For this purpose, a final conjugation step in the covalent approach (Figure 1: PS1D to PS1R) was carried out to evaluate the accessibility of amino groups on the polymersome surface of

between 4.5 and 7.75 ppm (Figure S13). However, the relatively high molecular weight of the copolymer leads to lower intensities of NVOC protons in the 1H NMR spectrum, which is in agreement with previous studies.6,39 This situation prohibited us from quantifying the conjugation yield precisely. Thus, we conducted another model study by applying the same aqueous click conditions to a short azide-terminated PEG molecule (N3-PEG60-OH, see SI). After successful covalent attachment of the photoactive groups to the polymersomes, we estimated the conjugation yield (conversion of PS2 into PS2C) as 26.5%. PS2C polymersomes were used here rather than PS1C polymersomes to obtain a more precise value by excluding the BMA contribution to the absorbance spectra. At first glance, this conjugation efficiency might be considered poor. However, almost half of the azide moieties are placed in the interior of the bilayer membrane and not available for the reaction.6,32,37 This implies that approximately 53% of azide moieties on the outer surface of polymersomes were converted to NVOC groups. By assuming the same conversion behavior for the conjugation step of PS1 into PS1C polymersomes ad well, the starting point for all further conjugation steps (Figure 1: PS1C to PS1D and PS1D to PS1R) in the covalent approach can be clarified. Thus, from this postulation, the resulting multifunctional polymersome system PS1C consists of approximately 7.7 mol % of NVOC, 9.2 mol % of adamantane, and 21.3 mol % of azide functionalities (Table 1). A noteworthy point here is that the 7.7 mol % of NVOC value depicts the modification only at the outer surface of PS1C polymersomes, whereas the other two values represent the H


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Figure 6. (a) UV−vis analysis to show sequential conjugation of the cyclodextrin molecule to rhodamine B NCS-modified polymersomes (PS1R) by host−guest interactions. (b) UV−vis spectra of Cy7-CD-containing polymersomes with (PS1H, PS1) and without (PS0) adamantane groups on their surface to show the conjugation triggered by host−guest interactions.

amine surface groups (Table 1). It is also noteworthy to point out that the amount of freely accessible amine groups on the surface can be tuned by UV exposure, which can lead to a controllable surface functionalization. In addition, cryo-TEM micrographs of PS1R polymersomes verify that the vesicles preserve their shape without any disassembly over the whole sequential conjugation steps as indicated previously for PS1D polymersomes (Figure 5d−g). Subsequent Noncovalent Conjugation through Adamantane−β-Cyclodextrin Host−Guest Interaction on the Polymersome Surface (PS1R to PS1H). In the last conjugation step, the accessibility of the adamantane functionality on the PS1R polymersome surface was studied in a noncovalent approach by the formation of host−guest inclusion complexes with β-cyclodextrin (β-CD) molecules. It is widely reported in the literature that adamantane groups tightly fit into the cavity of β-CD molecules, showing perfect host−guest interactions with a high association constant between 104 to 105 M−1.56,57 For this noncovalent conjugation approach on the PS1R surface, β-CD molecules were modified with a watersoluble Sulfo-Cyanine7 (Cy7) dye to monitor the host−guest inclusion complexation by UV−vis spectroscopy. From Figure 6a, it is apparent that the absorption band in the range of 600− 850 nm demonstrates the attachment of Cy7-labeled β-CD molecules (β-CD-Cy7) to adamantane groups in PS1H. For understanding this conjugation behavior in detail, the same process was carried out under identical conditions by using PS0 polymersomes without any adamantane groups (Figure 6b). This negative control experiment showed that PS0 polymersomes also interact to some extent with β-CD-Cy7 molecules. A possible explanation is that PEG chains in the polymersome corona of PS0 might thread into β-cyclodextrin cavities and form inclusion complexes. Various groups reported such behavior between PEG and α-cyclodextrin molecules.58,59

PS1D. Thus, rhodamine B isothiocyanate (RhB-NCS) was selected as a photostable and high quantum yielding fluorescent dye over a broad pH range53 to be reacted with the released NH2 groups of PS1D vesicles after UV exposure (Figure 5a). Next, an extensive purification step by dialysis was carried out to remove the unreacted reagent. As shown in Figure 5b, there is no change in the characteristic absorption bands of RhB-NCS dye (λmax = 555 nm) after 3 days of dialysis at basic conditions. This clearly indicates the successful conjugation of RhB-NCS molecules on the polymersome surface of PS1D. However, a possible physical interaction between RhB-NCS dye and the vesicle membrane might also contribute to the attachment.54 For investigating this possibility, the same process under identical conditions was applied to PS1 polymersomes. As shown in Figure 5b, NH2-functionalized polymersomes (PS1D) had approximately 1.4 times higher amount of RhB-NCS molecules conjugated than the ones without NH2 groups (PS1, control). This difference clearly confirms the successful covalent conjugation as well as the availability of the amine groups on the polymersome surface liberated after the photocleavage process of PS1C. We defined the absorbance difference of PS1R and the control experiment at λmax = 555 nm as a measure for the covalent attachment of the RhB-NCS dye (Figure 5b, A555 of PS1R = 0.893, A555 of PS1 = 0.655). This value was used in further calculations together with the measured molar extinction coefficient of RhB-NCS to estimate the conjugation efficiency of amino groups in polymersome PS1D (Table 1). This leads to a value of approximately 56.7% of amino groups on the surface of PS1R that reacted with the RhB-NCS dye. As indicated above, functional groups on the polymersome surface were placed as antennae with a long PEG segment to prohibit the concealment and steric effects of the hydrophilic corona.6,55 This high conjugation efficiency supports the argument of freely accessible and very reactive I


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Figure 7. Reversible swelling−shrinking of PS1C (a) and PS1D (b) polymersomes upon changes in pH value.

Figure 8. (a) Schematic overview of the Dox-encapsulated PS1C and PS1D formation. In vitro release of doxorubicin from PS1C-Dox polymersomes (b) and PS1D-Dox polymersomes (c) at 37 °C in different pH media.

Another reason might be related to the negatively charged sulfonate groups in the Cy7 dye. Although the membrane of the polymersome at pH 8 is mainly unprotonated, zeta potential results (Figure 2) outline a slight positive charge (ζpotential of approximately +5 mV). This can induce a weak interaction between sulfonate moieties and the PDEAEM groups of the membrane. Thus, it is assumed that this unexpected slight β-cyclodextrin binding to PS0 polymersomes without adamantane units is caused by the combination of both kinds of interactions. However, a comparison of UV−vis spectra of PS1H and PS0 polymersomes (Figure 6b) clearly

shows that the adamantane groups enhance the binding of CDCy7 molecules on the polymersome surface in PS1H significantly. Although this nicely proves the successful host− guest inclusion complexation of the β-cyclodextrin-modified dye by the adamantane groups on the polymersome surface (A775 of PS1H = 1.80, A775 of PS0 = 1.35), an additional investigation was performed on PS1 polymersomes. Our concern was to distinguish if there were any other interactions between cyclodextrin molecules and free functional groups on PS1H polymersomes (NVOC: 3.2 mol %, NH2: 1.9 mol %). As can be seen in Figure 6b, UV−vis spectra of polymersomes J


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The UV−vis monitoring of the vesicles before and after this cleavage process shows that doxorubicin molecules maintain their stability with only a small absorbance drop during the irradiation (Figure S19). This was an important issue for us to be able to truly analyze and interpret the release data. In addition, the DLS data of the PS1-Dox, PS1C-Dox, and PS1DDox reveal that doxorubicin incorporation did not make a notable change in the size of the vesicles in which the average diameters are 124.2 ± 1.08, 115.4 ± 1.01, and 119.4 ± 1.8 nm, respectively (Figure S20). Eventually, in vitro doxorubicin release from PS1C-Dox and PS1D-Dox was studied at pH 5 and 7.4 under physiological temperature (T = 37 °C). As seen in Figure 8, both of the polymersome systems exhibit a similar release profile. Dox was released to approximately 53% within 140 h at pH 5 from PS1C-Dox polymersomes, whereas for PS1D-Dox, the release was determined as approximately 51% under the same conditions. Although the membrane of PS1DDox became more compact due to additional UV irradiation, the pores induced by repelling of the polymer chains in the acidic state were sufficient for efficient drug release. Moreover, the closed state of the membrane decreased the undesired Dox release by a considerable amount at pH 7.4 to approximately 20% from PS1C-Dox and 26% from PS1D-Dox within 140 h (Figure 8b, c). Both of these results confirm our claim regarding tunable membrane permeability and reveal the potential of this multifunctional and dual responsive nanovesicle for various biomedical applications.

having only adamantane groups (A775 of PS1 = 1.87) showed a similar trend as for PS1H. This supports our claim that binding of β-cyclodextrin molecules to the PS1H surface is mainly triggered by the interaction with adamantane groups. To determine the efficiency of this conjugation, the same methodology was used as that for the rhodamine attachment step. The absorbance difference of PS1H and PS0 polymersomes at λmax = 775 nm was assigned as the value of the noncovalent conjugation, which was then used together with the measured molar extinction coefficient of CD-Cy7 molecules (Figure S18b). Finally, this calculation shows that 58.2% of adamantane groups on the outer shell of PS1H polymersomes were conjugated to the β-cyclodextrin molecules. Doxorubicin Encapsulation and pH-Triggered Release after Surface Modification of Polymersomes (PS1C-Dox, PS1D-Dox). In the final part, drug release experiments were carried out to demonstrate pH-controlled permeability of the established polymersome system. Here, doxorubicin hydrochloride (Dox) was selected as a model drug, which has a common clinical use in cancer chemotherapy.6−8 Because there is a relationship between permeability and swelling power caused by the repelling of the polymer chains in acidic conditions, we first investigated this phenomenon for PS1C (after the click reaction) and PS1D (after the photocleavage process) polymersomes as it was similarly performed for PS1. Figure 7 outlines that both polymersomes keep their stable nature for 5 cycles of swelling and shrinking experiments under acidic and basic states. Another essential result here is that there is almost no variation in the overall size increase of PS1C, the so-called swelling power, in comparison to PS1 polymersomes (Figure 2a). However, as expected, irradiation during the photocleavage process increased the cross-linking degree of the membrane, which causes lower swelling in the acidic state. Although the cross-linking degree can be tuned according to the needs of the application,19 we chose to examine the doxorubicin release on PS1C and PS1D polymersomes (Figure 8) to determine the feasibility of our multifunctional system from a drug delivery perspective. There are two common ways of cargo encapsulation for the pH responsive vesicles in which the cargo can be loaded during self-assembly (preloading) or afterwards (postloading). Herein, we chose the preloading approach to avoid any possible Dox trapped in the outer region of the polymersome membrane, which can inhibit effective drug encapsulation. In line with this, we also aimed to investigate the compatibility of our sequential postconjugation approach with the presence of a carrier. Thus, Dox encapsulation was performed before surface modification of the PS1 polymersomes during their formation, as illustrated in Figure 8a. After the self-assembly and cross-linking of the PS1-Dox polymersomes, an extensive purification using a hollow fiber filtration (HFF) system was carried out to remove all nonencapsulated Dox molecules. As a next step, the outer shell of the PS1-Dox polymersomes were modified with NVOC moieties by click chemistry as performed previously for the polymersomes without Dox (PS1) (Figure 1). Before the proceeding release study, any possible Dox leakage was also monitored during purification of NVOC moieties. There was only a slight Dox (15.5 ± 2.04%) leakage within 2 days of intensive dialysis at pH 7.4 (Figure S19). Subsequently, a certain amount of PS1C-Dox polymersomes was taken and irradiated for 600 s to cleave the NVOC moieties and finally to obtain PS1D-Dox polymersomes.

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CONCLUSIONS In the present work, we demonstrated the sequential postsurface functionalization of pH-sensitive and photo-crosslinked polymersomes with multiple reactive groups starting from azide and adamantane moieties. This presents the first example of heterofunctionalization of polymersomes suitable for drug encapsulation and release by applying several sequential reactions, implying covalent and noncovalent binding motifs in combination with a highly robust and responsive polymer membrane behavior. The sequential postconjugation of polymersomes started with a covalent approach (3 serial conjugation steps starting from azide units) and was finalized by a noncovalent approach for host−guest interactions of adamantane units with βcyclodextrin molecules. For the fabrication of multifunctionalized polymersomes, three amphiphilic block copolymers comprising adamantane, azide, and methoxy end groups at their hydrophilic PEG chain were synthesized. To guarantee the accessibility of functional units in sequential postmodification steps, the hydrophilic length of the azide- and adamantane-terminated block copolymers was designed to be longer than in the methoxy-terminated block copolymer. Moreover, these heterofunctionalized polymersomes, fabricated by three block copolymers, possess reversible swelling (pH 5) and shrinking (pH 10) behavior after photo-cross-linking the membrane. This in turn leads to controlled membrane permeability as demonstrated by doxorubicin encapsulation and further release experiments at different physiological conditions. In line with this, photo-cross-linked polymersomes show remarkable pH stability and preserve their vesicular shape over a wide range of pH values and under all postmodification steps. One key characteristics of the sequential postfunctionalization of the polymersome surface is the covalent conjugation of a prominent photoremovable compound, NVOC-protected K


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(6) Yassin, M. A.; Appelhans, D.; Wiedemuth, R.; Formanek, P.; Boye, S.; Lederer, A.; Temme, A.; Voit, B. Overcoming Concealment Effects of Targeting Moieties in the PEG Corona: Controlled Permeable Polymersomes Decorated with Folate-Antennae for Selective Targeting of Tumor Cells. Small 2015, 11, 1580−1591. (7) Ahmed, F.; Pakunlu, R. I.; Brannan, A.; Bates, F.; Minko, T.; Discher, D. E. Biodegradable Polymersomes Loaded with both Paclitaxel and Doxorubicin Permeate and Shrink Tumors, Inducing Apoptosis in Proportion to Accumulated Drug. J. Controlled Release 2006, 116, 150−158. (8) Onaca, O.; Enea, R.; Hughes, D. W.; Meier, W. StimuliResponsive Polymersomes as Nanocarriers for Drug and Gene Delivery. Macromol. Biosci. 2009, 9, 129−139. (9) Renggli, K.; Baumann, P.; Langowska, K.; Onaca, O.; Bruns, N.; Meier, W. Selective and Responsive Nanoreactors. Adv. Funct. Mater. 2011, 21, 1241−1259. (10) Peters, R. J. R. W.; Marguet, M.; Marais, S.; Fraaije, M. W.; van Hest, J. C. M.; Lecommandoux, S. Cascade Reactions in Multicompartmentalized Polymersomes. Angew. Chem., Int. Ed. 2014, 53, 146−150. (11) Gaitzsch, J.; Appelhans, D.; Wang, L.; Battaglia, G.; Voit, B. Synthetic Bio-nanoreactor: Mechanical and Chemical Control of Polymersome Membrane Permeability. Angew. Chem., Int. Ed. 2012, 51, 4448−4451. (12) Grzelakowski, M.; Onaca, O.; Rigler, P.; Kumar, M.; Meier, W. Immobilized Protein−Polymer Nanoreactors. Small 2009, 5, 2545− 2548. (13) Grzelakowski, M.; Onaca, O.; Rigler, P.; Kumar, M.; Meier, W. Immobilized Protein−Polymer Nanoreactors. Small 2009, 5, 2545− 2548. (14) Torchilin, V. P. Multifunctional nanocarriers. Adv. Drug Delivery Rev. 2012, 64, 302−315. (15) Le Meins, J. F.; Sandre, O.; Lecommandoux, S. Recent Trends in the Tuning of Polymersomes’ Membrane Properties. Eur. Phys. J. E: Soft Matter Biol. Phys. 2011, 34, 1−17. (16) Savić, R.; Azzam, T.; Eisenberg, A.; Maysinger, D. Assessment of the Integrity of Poly(caprolactone)-b-poly(ethylene oxide) Micelles under Biological Conditions: A Fluorogenic-Based Approach. Langmuir 2006, 22, 3570−3578. (17) Du, J.; Armes, S. P. pH-Responsive Vesicles Based on a Hydrolytically Self-Cross-Linkable Copolymer. J. Am. Chem. Soc. 2005, 127, 12800−12801. (18) Gaitzsch, J.; Appelhans, D.; Grafe, D.; Schwille, P.; Voit, B. Photo-Crosslinked and pH Sensitive Polymersomes for Triggering the Loading and Release of Cargo. Chem. Commun. 2011, 47, 3466−3468. (19) Yassin, M. A.; Appelhans, D.; Mendes, R. G.; Rümmeli, M. H.; Voit, B. pH-Dependent Release of Doxorubicin from Fast PhotoCross-Linkable Polymersomes Based on Benzophenone Units. Chem. Eur. J. 2012, 18, 12227−12231. (20) Blanazs, A.; Massignani, M.; Battaglia, G.; Armes, S. P.; Ryan, A. J. Tailoring Macromolecular Expression at Polymersome Surfaces. Adv. Funct. Mater. 2009, 19, 2906−2914. (21) Qin, S.; Geng, Y.; Discher, D. E.; Yang, S. TemperatureControlled Assembly and Release from Polymer Vesicles of Poly(ethylene oxide)-block- poly(N-isopropylacrylamide). Adv. Mater. 2006, 18, 2905−2909. (22) Napoli, A.; Valentini, M.; Tirelli, N.; Muller, M.; Hubbell, J. A. Oxidation-Responsive Polymeric Vesicles. Nat. Mater. 2004, 3, 183− 189. (23) Krack, M.; Hohenberg, H.; Kornowski, A.; Lindner, P.; Weller, H.; Förster, S. Nanoparticle-Loaded Magnetophoretic Vesicles. J. Am. Chem. Soc. 2008, 130, 7315−7320. (24) Cabane, E.; Malinova, V.; Menon, S.; Palivan, C. G.; Meier, W. Photoresponsive Polymersomes as Smart, Triggerable Nanocarrier. Soft Matter 2011, 7, 9167−9176. (25) Egli, S.; Schlaad, H.; Bruns, N.; Meier, W. Functionalization of Block Copolymer Vesicle Surfaces. Polymers 2011, 3, 252−280.

amine groups, by azide−alkyne click reaction. This directly induced light responsiveness to provide selectively free amine functionalities on the surface of polymersomes upon UV exposure. The presence of free amino groups can be used for further postconjugation steps as proven in our study by introducing dye molecules. Moreover, the controlled photochemical reaction can be performed to tune the amount of amino groups by simply varying the irradiation time. This also enables us to retain the off-state of NVOC-protected amine groups unless they are triggered externally. Thus, it was quantitatively proven that all functional groups were accessible and highly reactive to fabricate heterofunctionalized polymersome surfaces with different interaction and reactivity properties. Overall, these multifunctional polymersomes, possessing various reactive groups as well as a pH- and light-responsive nature, exhibit new opportunities for the enhancement of specific targeting and the establishment of multicompartmentalized systems in drug delivery and synthetic biology but also for future applications in “Lab-on-a-Chip” devices.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.5b05016. All synthetic procedures of the block copolymers, characterization methods, and further experimental procedures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Funding

This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) SPP 1327 Project and excellence cluster “cfaed” at TU Dresden. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Hartmut Komber for help with NMR measurements and analysis, Dr. Mikhail Malanin for performing IR measurements, Anja Caspari for conducting zeta potential measurements, and Christina Harnisch for carrying out SEC measurements.

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REFERENCES

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