Stimuli-Responsive Polymersomes for Biomedical Applications

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Stimuli-Responsive Polymersomes for Biomedical Applications Xiuli Hu,†,‡ Yuqi Zhang,† Zhigang Xie,‡ Xiabin Jing,‡ Adriano Bellotti,†,∥ and Zhen Gu*,†,§,∥

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Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United States ‡ State Key Laboratory of Polymer Chemistry and Physics, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Jilin 130022, People’s Republic of China § Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States ∥ Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States ABSTRACT: Polymersomes, the structural analogues of liposomes, are hollow structures enclosed by a bilayer membrane made from amphiphilic copolymers. Polymersomes have been proposed to mimic the structure and properties of cellular membranes and viral capsids. Excellent robustness and stability, chemical versatility for tunable membrane properties and surface functionalization make polymersomes attractive candidates for drug delivery, diagnostic imaging, nanoreactor vessels, and artificial organelles. In further biomimetic strategies, stimuli-responsive polymersomes that can recognize various external physical or internal biological environmental stimuli and conduct “on demand” release in dose-, spatial-, and temporal-controlled fashions have been widely developed. This Perspective focuses on recent advances in stimuli-responsive polymersomes and their potential biomedical applications. Representative examples of each stimulus, the advantages and limitations of different strategies, and the future opportunities and challenges are discussed.

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INTRODUCTION Molecular self-assembly is ubiquitous in nature and underlies the construction of structures necessary for life.1,2 Understanding and mimicking nature’s mechanisms, especially molecular self-assembly, has long been of particular interest for the creation of artificial nanostructures.3,4 Among the obtained various morphologies of the assemblies,5 lipid vesicles and polymersomes have received considerable attention for their morphological similarities to cellular membranes and viral capsids.6,7 Lipid vesicles, also called liposomes, are self-assembled from low-molecular-weight amphiphilic lipids.8 Polymersomes, also known as polymeric vesicles, are self-assembled from amphiphilic block or graft copolymers to form hollow structures surrounded by a polymeric bilayer membrane or complicated interdigitated and amphiphilic membrane structures.9,10 All of these structures consist of an aqueous interior. However, the molecular weights of lipids are generally less than 1 kDa, while those of block copolymers can be up to 100 kDa.11 Accordingly, the membranes of polymersomes may be up to 10-fold thicker than those of liposomes, which remarkably enhances the robustness and mechanical and chemical stability of polymersomes.11 This unique property of polymersomes has attracted research for extended circulation and prevention of premature drug release, making polymersomes attractive candidates for various applications in drug delivery, gene therapy, and diagnostic imaging.12−14 Moreover, the hollow bilayer or compartmentalized structure of polymersomes allows © 2017 American Chemical Society

for encapsulation of both hydrophobic and hydrophilic agents. Anticancer drugs, DNA, RNA, and vaccines have all been successfully encapsulated in polymersomes.15 Also, the properties of polymersomes can be extensively tailored and modified using the high flexibility and customizability of block copolymers. An impressive library of polymersomes with diverse sizes, architectures, surface properties, membrane properties such as permeability, and chemical functionalities has been constructed to date.16,17 For example, surface functionalization can enable polymersome transport through biological barriers to increase their availability at the target site, thus optimizing their pharmacokinetics and biodistribution of a drug. 18,19 In other biomimetic systems and advanced biomedical applications, polymersomes serve as compartments for in situ reactions at the nanoscale, such as nanoreactors, artificial organelles, and cell mimics.20,21 Research over the last two decades has yielded significant progress in the development of polymersomes since they were first reported by Meijer’s group22 and Eisenberg’s group23 in 1995. The physicochemical characteristics, self-assembly theory, and preparation methods of polymersomes as well as their application in drug delivery have been reviewed in several papers.5,15,24−28 For example, Santos and co-workers composed a review of the principles of block copolymer self-assembly, Received: November 17, 2016 Revised: January 27, 2017 Published: February 17, 2017 649

DOI: 10.1021/acs.biomac.6b01704 Biomacromolecules 2017, 18, 649−673

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BIOLOGICAL-STIMULI-RESPONSIVE POLYMERSOMES pH-Responsive Polymersomes. pH-responsive polymersomes are among the most studied stimuli-responsive systems because of the presence of physiological pH gradients within the body. For example, the extracellular pH of tumor and inflammatory tissues (pH ∼ 6.5−7.2) is slightly lower than that of normal tissues and blood (pH ∼ 7.4). The pH is even lower in endosomes (pH 5.5−5.0) and lysosomes (pH 4.5− 5.0).30,38,39 This physiological pH gradient makes pHresponsive polymersomes ideal candidates as drug delivery carriers, which have been widely exploited to deliver drugs to target locations, including intracellular compartments, specific organs, or microenvironments associated with certain pathological situations.40 Such pH-responsive polymersomes are generally constructed by incorporating acid-cleavable bonds38,41,42 or ionizable groups into the block copolymer43,44 or by directly forming polyionic complexes (PICsomes) via electrostatic interactions.45,46 The typical polymer structures used for pH-sensitive polymersomes are listed in Table 1A. Structures with Acid-Cleavable Bonds. In the earliest and most straightforward examples of pH-responsive polymersomes, a hydrolysis-susceptible aliphatic polyesters such as poly(lactic acid) (PLA)47 or poly(ε-caprolactone) (PCL)48 was used as the hydrophobic block. Discher and co-workers demonstrated pH-triggered hydrolytic degradation of the block copolymers, as evidenced by the morphological transitions of the polymersomes to micelles in releasing their cargoes (Figure 2).6,49,50 In vivo experiments showed tumor growth arrest and shrinkage after a single intravenous injection of polymersomes loaded with paclitaxel (TAX) and doxorubicin (Dox).51 Another example was an amphiphilic triblock copolymer synthesized by sequential thiol−acrylate Michael addition reactions with a main chain that is periodically segmented by an acid-labile β-thiopropionate functional group.52 The β-thiopropionate linker can be selectively hydrolyzed under mildly acidic conditions (pH 5.5), resulting in the sustained release of encapsulated guest molecules. To date, numerous acid-cleavable linkers, including hydrazone,53 imine,54 ortho ester,44,55 and acetal,38,41,56−58 have also been investigated for the preparation of polymersomes with tunable degradation kinetics. The acid-cleavable linkers can be integrated into the main chain or the pendant chains of the block copolymer. Zhong and co-workers synthesized pHsensitive polymersomes based on a diblock copolymer of poly(ethylene glycol) (PEG) and poly(2,4,6-trimethoxybenzylidenepentaerythritol carbonate) (PTMBPEC).56,58 The cyclic benzylidene acetal linker present in the pendant chains is highly sensitive to acidic environments and prone to fast hydrolysis at a mildly acidic pH, resulting in swelling and disassembly of the polymersomes. The groups of Zhong57 and Liu38 applied this cyclic benzylidene acetal to the side chains of a polymethacrylate-based block copolymer via reversible addition−fragmentation chain transfer (RAFT) polymerization. Hydrophobic and hydrophilic drugs were then simultaneously loaded into the hydrophobic membrane and aqueous core of the polymersomes, respectively, and released in a controllable and pHdependent manner. Targeting ligands were also introduced, and in vitro experiments indicated enhanced antitumor efficacy.57 Van Hest and co-workers synthesized polybutadiene-bpoly(ethylene glycol) (PBD-b-PEG) block copolymers in which the two segments were coupled via an acid-sensitive

preparation of polymersomes, and their applications for drug delivery, active targeting, and protocells.25 More recently, polymeric vesicles with bilayer or complicated interdigitated and amphiphilic membrane structures were reviewed by Du and co-workers, including the formation mechanisms, preparation methods, applications, and responsive behaviors.10 The latest emerging research focuses involve the design of stimuliresponsive “smart” polymersomes that can recognize some environmental stimuli and release a drug in dose-, spatial-, and temporal-controlled manners.29 A variety of stimuli-responsive polymersomes have been exploited to date.30−36 Zhong and coworkers previously reviewed advances in stimuli-sensitive polymersomes and highlighted their application for drug delivery.30 Li and Keller37 reviewed the chemical and physical structures of the copolymers forming the stimuli-responsive polymer vesicles and their effects on the controlled release of the encapsulated contents. The stimuli are typically classified into two groups:29 internal biological stimuli such as endolysosomal pH, redox potential, enzymatic activities, and monosaccharide concentration and external physical stimuli such as temperature, light, electric field, mechanical force, and ultrasound (Figure 1). Responsiveness to these stimuli can be

Figure 1. Schematic illustration of various stimuli-responsive polymersomes for different biomedical applications.

introduced in a drug delivery system by integrating intrinsically stimuli-responsive chemical groups into the block copolymer. The synthesized polymersomes are then capable of physical and chemical changes, such as swelling, membrane fusion, disassembly, and bond cleavage, in response to specific stimuli, thus subsequently leading to polymersome disruption and drug release. In this Perspective, we focus on recent advances in stimuli-responsive polymersomes from different aspects, including the mechanism and representative examples of each stimulus and the advantages and limitations of different strategies for their potential biomedical applications. Future opportunities and challenges regarding translation are also discussed. 650


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Table 1. Typical Polymer Structures or Reactions Used for Stimuli-Responsive Polymersomes Described in this Paper

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hydrazone moiety.53 Polymersomes composed of a mixture of stable and cleavable PBD-b-PEG were constructed by mixing PBD-b-PEG with an inert analogue. Ninety-five percent of peripheral PEG chains could be systematically removed by lowering the pH without disrupting the colloidal stability. Yang and co-workers prepared pH-sensitive polymeric vesicles by coassembling cholate-grafted poly(L-lysine) (PLys-CA) with an

amphiphilic PEG−Dox conjugate formed via an acid-labile benzoic imine bond.54 The acid-labile bond in the PEG−Dox conjugate resulted in pH-responsive membrane permeability and triggered dissociation of the polymersome following a drop in environmental pH. Structures with Ionizable Groups. pH-sensitive polymers with ionizable groups typically have weakly acidic groups such 657


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Tertiary-amine-containing methacrylate copolymers were another often-investigated system for which a host of excellent examples with varying pKa have been reported and recently reviewed by Liu and co-workers.59 Poly(2-(diisopropylamino)ethyl methacrylate) (PDPA),46 poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA),63,64 and their dimethyl analogue (PDMAEMA)65 have been extensively investigated as polymersome systems by the groups of Armes and Voit. For example, Armes and co-workers used zwitterionic poly(2(methacryloyloxy)ethylphosphorylcholine)-b-poly(2(diisopropylamino)ethyl methacrylate) (PMPC-b-PDPA) diblock copolymers to prepare stable vesicles at physiological pH (Figure 4).46 When the pH of the solution was lowered, a sharp morphological transition was observed as the vesicles dissolved into polymers as a result of protonation of the tertiary amine groups on the PDPA block, which is hydrophobic at physiological pH and hydrophilic in acidic solutions. PDPA has proven to be a perfect candidate for drug delivery applications because of its pKa (∼6.4). The polymersomes were stable in the bloodstream (pH ∼ 7.4), and the pH sensitivity of the PDPA block allowed immediate cargo release once the polymersomes entered the endolysosomal compartments.66 In a following study, PMPC-b-PDPA polymersomes were further employed for the efficient intracellular delivery of DNA,67,68 siRNA,69 antibodies,70 anticancer drugs,71,72 and fluorophores.66,73 Furthermore, the cellular uptake kinetics was effectively controlled by the size, surface chemistry, and topology of the polymersomes.68,74 The hollow and compartmentalized compartments in polymersomes are ideal nanoreactors and nanocapsules.75 Voit and co-workers employed block copolymers composed of pH-sensitive DEAEMA and a photo-cross-linking unit to prepare cross-linked polymersomes as nanoreactors.63,64,76 The resultant morphologically persistent polymersomes were used to encapsulate Dox.77−79 The release of the loaded anticancer drug Dox was controlled by modifying the cross-linking density of the polymersome and the pH of the environment (Figure 5).79 Folic acid was introduced as a targeting moiety for selective toxicity in tumor cells compared with healthy cells.78 Polypeptide-containing block copolymers are also among the earliest self-assembled polymers and have attracted considerable interest for their high biocompatibility, biodegradability, and complex secondary conformations.80,81 More importantly, the conformation of the peptide with ionizable side groups can be reversibly manipulated by environmental changes in pH, ionic strength, temperature, or solvent quality, which regulate the morphology of peptide-based nanoparticles.82 In addition, the ionizable groups can also be used to load oppositely charged drugs or bioactive macromolecules via electrostatic interactions.83 Polymersomes formed from polypeptide-based copolymers, also known as pepsomes, have been developed and tested for various biomedical applications.84 Cationic polypeptides such as PLys, polyhistidine (PHis), and polyarginine (PArg) as well as the anionic polypeptides poly(glutamic acid) (PGA) and poly(aspartic acid) (PAsp) are integrated with other polymers, including polypeptides, polyesters, and carbohydrates,84 to form amphiphilic polypeptides. Förster group85 and Lecommandoux group82,86 synthesized PGA- and PLys-based polypeptide block copolymers containing PBD,82,85,86 polyisoprene (PI),87 or poly(trimethylene carbonate) (PTMC).83,88 They investigated the pH sensitivity of the nanoparticles that results from the polyelectrolyte corona and secondary structure of PGA or PLys in the block copolymers.

Figure 2. PEG−PLA-based polymersome self-assembly, degradation, and drug release. (A) Cryo-TEM images of empty aggregates. Hydrolysis of PLA in the vesicle core triggers the growth of pores and conversion of vesicles into wormlike micelles and spheres. Scale bars are 100 nm. (B) In vitro release and leakage of doxorubicin (Dox) and paclitaxel (TAX) from degradable and nondegradable polymersomes. Reprinted with permission from ref 50. Copyright 2006 Elsevier.

as carboxylic or sulfonic acids (i.e., polyacids) and/or weak basic groups such as primary, secondary, or tertiary amine groups (i.e., polybases).30,37,59 Their sensitivity can come from changes in conformation or solubility in response to variation in environmental pH via ionization (protonation or deprotonation). Eisenberg’s group pioneered the polyacid-based systems in their early work on mapping the phase diagram of the charged block copolymer polystyrene-b-poly(acrylic acid) (PAA-b-PS) in dilute organic or aqueous solutions.23 Liu and Eisenberg60 later observed the pH-triggered morphological change of the triblock copolymer poly(acrylic acid)-bpolystyrene-b-poly(4-vinylpyridine) (PAA-b-PS-b-P4VP) from vesicles to solid spherical aggregates and then back to vesicles based on the different repulsive interactions within the PAA or P4VP corona at various pH. Discher and co-workers investigated salt- and pH-induced morphological changes in the block copolymer poly(acrylic acid)-b-polybutadiene (PAAb-PBD).61 Chiu et al.62 reported multivesicles equipped with pH-responsive transmembrane channels based on the random polymer poly(acrylic acid-co-distearin acrylate) (poly(AA-coDSA)). At a pH of 5.0, the channels were closed because of hydrogen bonds and hydrophobic association of deionized AA. When the pH was increased to 6.5, ionization of AA occurred, leading to disruption of the hydrogen bonds and hydrophobic association and the creation of permeable channels, as shown in Figure 3. The pH-induced on/off process was found to be reversible, and the channels were accessible to both small molecules such as calcein and larger cargoes such as hemoglobin. 658


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Figure 3. (A) Illustration of multivesicular assemblies equipped with pH-responsive transmembrane channels from a double emulsion of poly(AAcco-DSA). (B) Confocal laser scanning microscopy (CLSM) images of Nile red-stained vesicle suspensions (a) with the addition of calcein at pH 5.0 (calcein could not enter the vesicle), (b) after pH adjustment to 8.0 (calcein diffused into the vesicle), and (c) after replacement with fresh buffer (pH 5.0) (calcein was confined within the vesicle). Reprinted with permission from ref 62. Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 4. A. Formation of PMPC25-b-PDPA120 block copolymer vesicles. (B) (a) Variation of the hydrodynamic diameter of self-assembled vesicles in aqueous solution vs solution pH (the initial copolymer concentration at pH 2 was 1.0 g/L). (b) Doxorubicin (Dox) elution profile from PMPC25b-PDPA120 vesicle solution (pH 7.5 saline buffer at 20 °C). Reprinted from ref 46. Copyright 2005 American Chemical Society.

increasing temperature.83 This system was also investigated for both therapeutic and diagnostic applications by encapsulating ultrasmall superparamagnetic iron oxide (γ-Fe2O3) nano-

The anticancer drug Dox was loaded in the polymersomes with high loading efficiency and high stability at room temperature, and the drug release rate increased at acidic pH or with 659


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Figure 5. (A) Schematic illustration of shape-persistent polymersomes with pH-governed membrane permeability and controlled release of the anticancer drug doxorubicin. (B) TEM images of polymersomes irradiated for 5 min at pH 10 (left) and at pH 2 (right). Reprinted with permission from ref 79. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kataoka and co-workers prepared a series of stable polymersomes by simply mixing a pair of oppositely charged block copolymers that contain a PEG block and an ionic block prepared from aniomers and catiomers, respectively, in an aqueous medium.90−92 The resultant polymersomes were termed polyion complexes (PICsomes). PICsomes manifested several advantages compared with traditional polymersomes, such as no requirement for organic solvent and easy encapsulation of water-soluble macromolecules. Kataoka’s group prepared PICsomes with precise control of the size, distribution, and structure and obtained stable polymersomes with tunable membrane permeability by chemical cross-linking of the PIC layer.91,93 Highly sensitive MRI contrast agents were synthesized by encapsulating SPIO NPs into the PICsomes, and this contrast was evaluated for detection of small tumors and early diagnosis of cancer in mice subcutaneously grafted with colon-26 tumor cells.94 On the other hand, myoglobin (Mb) was encapsulated into poly(ethylene glycol)-b-poly(α,βaspartic acid) (PEG-b-P(Asp)) and poly(ethylene glycol)-bpoly((5-aminopentyl)-α,β-aspartamide) (PEG-b-P(Asp-AP)) PICsomes to form oxygen carriers with potential use in vivo as shown in Figure 7.95 Reduction-Responsive Polymersomes. Reduction-responsive polymersomes have attracted considerable attention and have been widely investigated for triggered intracellular

particles (USPIO NPs) and Dox. The encapsulated USPIO NPs may also be used to trigger drug release under a magnetic ́ ndez and field.89 In another interesting paper, Rodriguez-Herná 45 Lecommandoux reported reversible polymersomes assembled from the zwitterionic diblock copolymer PGA-b-PLys as a function of pH in water (Figure 6).

Figure 6. Schematic representation of the self-assembly of the diblock copolymer PGA15-b-PLys15 into vesicles. Reprinted from ref 45. Copyright 2005 American Chemical Society. 660


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Figure 7. (A) Reversible Mb oxygenation inside the PICsome self-assembled from a pair of oppositely charged block ionomers. (B) Cross-sectional image of TRITC-Mb loaded in PICsomes observed by CLSM. (C) Changes in the absorbance at 434 nm of the Mb-PICsome upon alternating introduction of O2 and Ar gas to the solution, indicating the reversible generation of oxy-Mb and deoxy-Mb. Reprinted with permission from ref 95. Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

reversibly cross-linked temperature-responsive polymersome self-assembled from the triblock copolymer poly(ethylene oxide)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide) (PEO-b-PAA-b-PNIPAM) and further cross-linked using cystamine via carbodiimide chemistry.102 The cross-linked polymersomes showed remarkable stability when dissolved or exposed to organic solvents, high salt conditions, and temperature variations, and the polymersomes could rapidly dissociate under reductive conditions, quickly releasing the preloaded sample protein, FITC-dextran. Reduction- and pHsensitive cross-linked polymersomes based on the PEG-b-PAAb-PDEAEMA triblock copolymer cross-linked with cystamine were also reported by the same group. These polymersomes showed potential for efficient intracellular delivery of proteins.110 In another report, the same group prepared reversibly stabilized multifunctional dextran nanoparticles based on dextran−lipoic acid derivatives (Dex-LAs) (Figure 8)111 in which the lipoic acid (LA) is responsible for the crosslinking and the reduction response.112 Multifunctional polymersomes were also prepared from poly(ethylene glycol)-bpoly(trimethylene carbonate-co-dithiolane trimethylene carbonate) (PEG-b-P(TMC-DTC)), in which the dithiolane ring, an analogue of lipoic acid, was introduced into the pendant chains of poly(trimethylene carbonate) and the cyclic peptide cNGQ was decorated on the surface.113 The obtained Dox-loaded, cNGQ-decorated polymersomes showed superior treatment of orthotropic human lung cancers compared with Dox. In contrast to the aforementioned chemical cross-linking, Li and co-workers reported reduction-responsive polymersomes based on the amphiphilic block copolymer PEG-SS-polyacrylate/ cholesterol (PAChol). 114 The reduction sensitivity was introduced by incorporating disulfide bridges between the hydrophilic PEG blocks and the hydrophobic PAChol blocks, while the stability was obtained by physical cross-linking of cholesterol.

release of various drugs due to the significant redox potential difference within different cell organelles and the cytosol.96−98 The tripeptide glutathione (GSH) is found to have a low concentration in plasma (typically 1−2 μM) and normal tissues (approximately 2−20 μM), while tumor tissues exhibit a 4-fold increase in GSH in tumor-bearing mice.99 In particular, the cytosol and cell nuclei have much higher concentrations of GSH (approximately 2−10 mM), which create a high intracellular redox potential.100 Disulfide bonds are readily known to be responsive to reduction and can be reduced to two thiols in the presence of various reducing agents, including GSH. Disulfide bonds are simply introduced into backbone of the copolymer or as the cross-linkers in the pendant chains.101−105 Cleavage of these bonds can induce disassembly of the polymersomes in reductive environments. Various reduction-responsive copolymers have been reported; however, there are far fewer studies involving reduction-responsive polymersomes. Hubbell and co-workers developed reductionsensitive polymersomes based on poly(ethylene glycol)-SSpoly(propylene sulfide) (PEG-SS-PPS). The prepared polymersomes were demonstrated to dissociate in the presence of intracellular concentrations of cysteine, releasing their cargoes within 10 min of exposure to cells.101 Li and co-workers reported polymer vesicles self-assembled from triblock copolymers of PEG and poly(ε-benzyloxycarbonyl-L-lysine) (PzLL) that were linked by two disulfide bonds (PzLL-SSPEG-SS-PzLL).106 Dox-loaded vesicles showed enhanced cell accumulation after incubation with HeLa cervical cancer cells upon exposure to GSH and were helpful in reversing drug resistance. Zhong and co-workers reported a series of reduction-sensitive biodegradable polymersomes based on hydrophilic PEG and hydrophobic PCL,107 PDEAEMA,108 and their copolymers when mixed.109 In order to improve the stability of polymersomes while maintaining their responsiveness, disulfide bonds can be introduced as cross-linkers. Zhong and co-workers reported a 661


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properties, and potential biomedical applications have been summarized in several recent reviews.116,126−130 This section summarizes the limited examples of oxidation-responsive polymersomes. Hubbell and co-workers reported the first example of using oxidative conversion to destabilize polymersomes.131 They synthesized the triblock copolymer poly(ethylene glycol)-bpoly(propylene sulfide)-b-poly(ethylene glycol) (PEG-b-PPS-bPEG), which could self-assemble into vesicles in aqueous solutions. The central-block hydrophobic sulfide moieties could be converted to sulfoxides and ultimately sulfones upon exposure to oxidative conditions, inducing the conversion of PPS from a hydrophobe to a hydrophile and the morphology change from stable vesicles to wormlike micelles to spherical micelles and ultimately to nonassociating unimolecular micelles. Glucose oxidase (GOx)/glucose was encapsulated in the vesicles, and glucose-triggered vesicle destabilization through the production of hydrogen peroxide was studied.34 In another system, Hubbell’s group investigated the oxidation sensitivity of polymersomes based on the block copolymer PEG-b-PPS132 and applied these polymersomes as a vaccine delivery platform by encapsulating antigen and adjuvant drugs for inducing cellmediated antigen-specific immune responses, as shown in Figure 9.133 Boronic esters have been widely investigated as oxidationresponsive materials for H2O2-induced degradation. In the pioneering work, Fréchet and co-workers reported nanoparticles self-assembled from phenylboronic ester-modified dextran that sensitively respond to H2O2 at concentrations as low as ∼1 mM.134 In another system, Liu and co-workers reported oxidation-responsive polymersomes based on two phenylboronic ester derivatives. 135 The obtained block copolymer could self-assemble into diverse aggregates, including spherical nanoparticles and polymersomes, in aqueous media. Oxidation responsiveness was observed when these aggregates were incubated with H2O2 under simulated biological conditions (∼1 mM H2O2) or in the cellular oxidative milieu inside live cells. Gu and co-workers also reported hypoxia vesicles136 and hypoxia/H2O2 dual-sensitive vesicles124 obtained by conjugating 6-(2-nitroimidazole) hexylamine to hyaluronic acid and conjugating (2-nitroimidazol-1yl)methanethiol to the block copolymer PEG-b-polyserine, respectively. The sensitivity of vesicles responding to the generated H2O2 and local hypoxia during glucose oxidation catalyzed by a glucose-specific enzyme was assessed. Under hypoxic conditions, the hydrophobic 2-nitroimidazol groups were converted to hydrophilic 2-aminoimidazole groups

Figure 8. Schematic illustration of the preparation and intracellular fate of reversibly stabilized, multifunctional dextran−lipoic acid nanoparticles. Reprinted with permission from ref 111. Copyright 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oxidation-Responsive Polymersomes. Reactive oxygen species (ROS)-responsive molecules are an emerging biomaterial in the field of internal biological stimuli.115 ROS generally include hydroxyl radical (OH·), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and superoxide (O2−), which are produced from several endogenous sources and serve crucial roles in physiological processes such as cellular signaling, apoptosis, cellular proliferation, and immune responses.116 However, overproduction of ROS may result from oxidative stress, a biological feature that is accompanied by various pathological disorders, including cancer, inflammation, infections, cardiovascular disease, and diabetes.117−120 Various probes have been developed for the detection or diagnosis of diseases involving oxidative stress.121,122 The abnormal redox states in tumor and inflammatory tissues make the pathological sites distinct from their surroundings and have been considered as targets for site-specific delivery of therapeutic and imaging agents. Oxidation-responsive polymers based on various motifs have been developed,123−125 and their synthesis, oxidative

Figure 9. (A) Schematic illustration of PEG-b-PPS block copolymer vesicles. (B, C) Bodipy-tagged PEG-b-PPS polymersomes were loaded with (B) green fluorescent protein or (C) calcein using thin-film rehydration. Reprinted with permission from ref 133. Copyright 2012 Elsevier. 662


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Figure 10. (A) Schematic illustration of the vesicular structure of MO65-b-(L0.5/F0.5)20 copolypeptides. (B) Enzyme-triggered release of probe molecules from methionine sulfoxide-containing vesicles. (a) Plot showing cumulative release of Texas Red-labeled dextran from MO65-b-(L0.5/F0.5)20 vesicles over time. Blue diamonds: MO65-b-(L0.5/F0.5)20 vesicles incubated with DTT at 37 °C for 16 h. Red squares: MO65-b-(L0.5/F0.5)20 vesicles incubated with DTT and methionine sulfoxide reductase A and B at 37 °C for 16 h. (b) Schematic illustration of the possible effect of enzymatic reduction of vesicle surface MO segments to M segments. Reprinted from ref 143. Copyright 2013 American Chemical Society.

served as the substrate for methionine sulfoxide reductase enzymes and could be converted to hydrophobic M segments, resulting in vesicle disruption and release of the cargo, as shown in Figure 10 and Table 1C. Glucose-Responsive Polymersomes. Glucose-responsive polymeric materials have attracted considerable attention in recen years because of their potential in the construction of closed-loop smart insulin delivery systems for the treatment of type-1 and advanced type-2 diabetes.144−146 Three typical strategies have been developed by incorporating different glucose-sensing moieties into polymersomes, including glucose oxidase (GOx), glucose-binding proteins, and boronic acids. The obtained matrix can undergo structural transformations, such as shrinking, swelling, and dissociation, regulated by glucose concentration changes, leading to glucose-stimulated insulin release136,147,148 or glucose-mediated membrane permeability.21 Typical polymer structures used for glucoseresponsive polymersomes are listed in Table 1D. Boronic acid-containing materials have been widely studied and used in the construction of glucose-responsive systems for insulin delivery because of their ability to form reversible complexes with 1,2-cis-diols of glucose.149,150 Kim et al. synthesized block copolymers of poly(ethylene glycol)-bpoly(styreneboroxole) (PEG-b-PBOx).151,152 By adjusting the degree of polymerization of the PBOx block, they obtained a variety of nanostructures, including spherical and cylindrical micelles and polymersomes. The obtained polymersomes exhibited monosaccharide-responsive disassembly in a neutralpH medium and were used to encapsulate insulin. Monosaccharide-regulated insulin release was investigated. Van Hest and co-workers reported polymersome nanoreactors with controllable permeability obtained by incorporating the glucose-responsive block copolymer poly(ethylene glycol)-bpoly(styreneboronic acid) (PEG-b-PSBA) with the conventional amphiphilic block copolymer poly(ethylene glycol)-bpolystyrene (PEG-b-PS).21 The water solubility of the PSBA block in PEG-b-PSBA increases when the boronic acid is ionized to the boronate in a high-pH medium or binds with sugar molecules, which promotes disassembly of polymersomes made of PEG-b-PSBA into dissolved block copolymers. Glucose-responsive systems with glucose-sensing moieties (GOx) are always integrated with other pH-, hypoxia-, or oxidation-responsive materials by taking advantage of the local acidic,147,153,154 hypoxic,124,136 or H2O2-containing34,123 environment generated in the enzymatic reaction. Gu and co-

catalyzed by nitroreductases, and meanwhile, the thioether linker served as a H2O2-sensitive moiety and was converted into a more hydrophilic sulfone by H2O2. This synergistic effect promoted the dissociation of the dual-sensitive vesicles to release the encapsulates. Selenium/tellurium-containing polymers have been widely investigated by Xu and co-workers.137,138 The redox response and self-assembly behaviors were summarized.130 Typical polymer structures used for reductionand oxidation-responsive polymersomes are listed in Table 1B. Enzyme-Responsive Polymersomes. Enzyme-responsive formulations have been widely exploited to achieve sensitive, selective, and efficient targeted delivery of therapeutic agents at specific sites with a specific enzyme.139 Heise and co-workers prepared poly(L-glutamic acid-co-alanine)-b-poly(n-butyl acrylate) (P(GA-co-Ala)-b-PBA) and poly(L-glutamic acid-coalanine)-b-polystyrene (P(GA-co-Ala)-b-PS) block copolymers with various quantities of Ala, which could be degraded by elastase and thermolysin.140 The corresponding biohybrid vesicles or micelles obtained showed varying degrees of enzyme responsiveness when exposed to elastase and thermolysin, and enzymatic degradation of parts of the polypeptide block resulted in particle destabilization. Liu and co-workers reported an enzyme-responsive vesicle with p-sulfonatocalix[4]arene (SC4A) as the macrocyclic host and natural enzyme-cleavable myristoylcholine as the guest molecule.141 The self-assembled vesicles exhibited highly specific and efficient responsiveness to cholinesterase, a key protein overexpressed in Alzheimer’s disease, indicating their great potential for use in the delivery of drugs for Alzheimer’s disease. Balasubramanian and co-workers prepared dextran vesicular nanoscaffolds based on polysaccharide and a renewable-resource alkyl tail, which were used for dual encapsulation of hydrophilic rhodamine B (Rh-B) and hydrophobic camptothecin (CPT).142 The aliphatic ester linkage connecting the hydrophobic tail with dextran was demonstrated to be cleaved by esterase under physiological conditions for fast release of CPT or Rh-B. In contrast to bond formation or cleavage in responsive materials, Deming and coworkers reported that oxidation/reduction reactions induced a hydrophobic/hydrophilic transition of the polymer, resulting in vesicle disruption and cargo release.143 They first prepared a fully hydrophobic precursor diblock copolypeptide, poly(Lmethionine)-b-poly(L-leucine-stat-L-phenylalanine), M 65-b(L0.5/F0.5)20, and its direct oxidation in water gave the amphiphilic MO derivative MO65-b-(L0.5/F0.5)20. The amphiphilic MO derivative self-assembled into vesicles in water, which 663


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Figure 11. Schematic illustration of the glucose-responsive insulin delivery system using hypoxia and H2O2 dual-sensitive polymersome-based vesicles (d-GRPs) loading microneedle-array patches. (A) Formation and mechanism of d-GRPs comprised of PEG-poly(Ser-S-NI). (B) Schematic of local inflammation induced by non-H2O2-senstive GRP-loaded microneedle-array patch, and schematic of d-GRP-loaded microneedle-array patch for in vivo insulin delivery triggered by a hyperglycemic state for potential prevention of the long-term side effect associated with inflammation. (C) TEM images of d-GRPs encapsulating insulin and enzyme pre- or postincubation with 400 mg/dL glucose for 20 min, 1 h, and 24 h. Scale bar is 100 nm. Reprinted from ref 124. Copyright 2017 American Chemical Society.

which changed the hydrophilic−lipophilic balance of the polymer and thus destroyed the vesicles. Recently, Gu and co-workers reported a biodegradable and biocompatible H2O2triggered glucose-responsive insulin delivery system by integrating H2O2-responsive polymeric vesicles with transcutaneous patches.123 The reported polymeric vesicles were self-assembled from phenylboronic ester-modified poly(ethylene glycol)-b-polyserine block copolymer, and their H2O2-responsive capability was evaluated. Gas-Responsive Polymersomes. Carbon dioxide, nitric oxide, oxygen, and hydrogen sulfide are typical biologically active gases that play important and fundamental roles in human biology. Recently, the concept of using these gases as “green” stimuli to construct responsive polymer assemblies has received increasing attention. Among them are CO2- or N2responsive polymersomes, in which inactive CO2 or N2 gas is used to regulate the self-assembly and disassembly of the aggregates (Table 1E).155−157 Yuan and co-workers reported a series of CO2-responsive polymersomes containing amidine moieties, which can be transformed into charged amidinium bicarbonate upon reaction with CO2, a reaction that is reversible upon exposure to argon.157 They synthesized the amphiphilic block copolymer

workers constructed self-regulated insulin delivery nanovesicles by encapsulating GOx and insulin in pH-sensitive polymersomes composed of PEG and ketal-modified polyserine.147 Glucose can passively transport across the bilayer membrane of the polymersome for oxidation into gluconic acid by GOx, thereby causing a decrease in local pH. The acidic microenvironment causes the hydrolysis of the pH-sensitive polymersome, which in turn triggers the release of insulin in a glucose-responsive fashion. In vivo studies demonstrated that the polymersome was highly biocompatible and effective in regulating blood glucose levels for a long period of time. The local hypoxic microenvironment caused by the enzymatic oxidation of glucose into gluconic acid in the hyperglycemic state was utilized by Gu and co-workers to construct a glucoseresponsive insulin delivery device. Hypoxia and H2O2 dualsensitive vesicles were integrated into a painless microneedlearray patch to realize fast insulin release and convenient administration124 (Figure 11). Hubbell and co-workers34 encapsulated GOx into oxidation-responsive vesicles selfassembled by the block copolymer poly(ethylene glycol-bpropylene sulfide) (PEG-b-PPS). The H2O2 generated in the enzymatic oxidation of glucose reacted with the PPS block, converting it into more hydrophilic sulfoxides and sulfones, 664


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Figure 12. (A) (a) Gas-switchable chemical structural change of the PEG-b-PAD block copolymer. (b) Self-assembly of the copolymer into polymersomes and reversible gas-controlled breathing behavior in aqueous media. (B) Illustration of polymersomes acting as size-selective nanoseparators upon modulation of the CO2 level. (C) (a−c) TEM images showing the morphological changes in the PEG-b-PAD polymersomes under various conditions: (a) no stimulus; (b) after 10 min of CO2 exposure; (c) after 30 min of CO2 exposure. Scale bars are 200 nm. (d) Dynamic light scattering data for the size changes of the PEG-b-PAD polymersomes: no stimulus (blue); after 10 min of CO2 (red); after 30 min of CO2 (green). (e) Polymersome size as a function of gas stimulation time. (f) Variation of the membrane thickness of the polymersomes upon CO2 addition. The polymer concentration in all of the experiments was 0.20 mg mL−1. Reprinted with permission from ref 157. Copyright 2013 WileyVCH GmbH & Co. KGaA, Weinheim.

PDEAEMA blocks always constituted the inner CO2-responsive part of the core. Diversiform CO2-controlled deformations, such as volume expansion of nanospheres, stretching of curly nanofibers, and compartmentalization of vesicles, were achieved through CO2-induced protonation of the pendant tertiary amine in the PDEAEMA segment. They reported two other CO2-responsive vesicles based on the block copolymers PDMA-b-PDEAEMA (PDMA = poly(N,N-dimethylacrylamide)) and PEO-b-P(DEAEMA-co-CMA) (CMA = coumarin) using the same mechanism.155 For PDMA-b-PDEAEMA vesicles, morphological changes ranging from expansion to complete dissociation were realized upon CO2 bubbling as a result of protonation of the DEAEMA units in the vesicle membrane. PEO-b-P(DEAEMA-co-CMA) vesicles containing cross-linked membranes formed by CMA dimerization underwent reversible expansion and contraction under alternating passage of CO2 and Ar in solution. Both systems were investigated as CO2-controllable drug release carriers. Yuan and co-workers further combined CO2-responsive PDEAEMA with temperature-responsive PNIPAM to obtain a CO2- and temperature-switchable “schizophrenic” block copolymer and realized micelle-to-unimer-to-vesicle morphological transition.162

poly(ethylene glycol)-b-poly((N-amidino)dodecylacrylamide) (PEG-b-PAD), which could self-assemble into vesicles in aqueous solution. In this reaction driven by CO2, the hydrophobic part of PAD in the vesicular wall is transformed from an unprotonated and entangled state (polyamidine) to a protonated and stretched state (polyamidinium), inducing selfexpansion of the vesicles (Figure 12). Reversible expansion and contraction cycles of these vesicles occur with alternating treatments of CO2 and Ar.158 By regulation of the CO2 stimulation time, the growth of polymersomes and membrane permeability can be tuned, which is useful for controlling cargo release and selective separation of different guest molecules. This system can also be used as a nanoreactor for enzymatic catalytic reactions. Yan and Zhao159 used the same mechanism to observe CO2-regulated self-assembly and shape transformation from microscopic tubules to submicroscopic vesicles and nanomicelles by modulation of the CO2 level. Zhao’s group also reported CO2-responsive glycopolypeptide vesicles assembled from two end-decorated biopolymers, dextran-βcyclodextrin (Dex-CD) and poly(L-valine)-benzimidazole (PVal-b-Bzl), which display a reversible assembly and disassembly process that can also be tuned by CO2, biomimicking virus capsids.160 Another class of CO2-responsive polymers are amine- or carboxylic acid-containing polymers. Yan and Zhao161 synthesized poly(ethylene oxide)-b-polystyrene-b-poly((2diethylamino)ethyl methacrylate) (PEO-b-PS-b-PDEAEMA) triblock copolymers. Three initial nanostructures, including spherical micelles, wormlike micelles, and vesicles, were obtained by varying the length of the PS block, while the

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EXTERNAL-STIMULI-RESPONSIVE POLYMERSOMES Temperature-Responsive Polymersomes. Temperature is a popular stimulus that has been widely used and studied in triggering polymersomes.163−165 Among various thermoresponsive polymers, block copolymers containing poly(N665


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Figure 13. (A) General procedure for the preparation of BSA-NH2/PNIPAAm proteinosomes. (a) Coupling of mercaptothiazoline-activated PNIPAAm polymer chains with primary amine groups of cationized BSA-NH2 to produce protein−polymer nanoconjugates (BSA-NH2/ PNIPAAm). (b) Interface assembly of proteinosome microcompartments in oil and their transfer into a bulk water phase. (B) Schematic illustration showing the procedure for cell-free gene expression of eGFP in proteinosomes. Reprinted with permission from ref 168. Copyright 2013 Macmillan Publishers Ltd.

PNIPAM to proteins to obtain thermally responsive microcompartments called “proteinsomes”,168 which have since been evaluated as synthetic protocells for guest molecule encapsulation, selective permeability, protein synthesis via gene expression, and membrane-gated internalized enzyme catalysis (Figure 13). Mann’s group demonstrated the thermally induced gating of membrane permeability to external substrates, which effectively creates an on/off switch for enzymatic reactions inside the microcompartments. Temperature responsiveness can be integrated with other functions to achieve cross-linked or dual/multiresponsive polymersome systems. McCormick and co-workers prepared temperature-responsive polymersomes based on poly(N-(3aminopropyl)methacrylamide hydrochloride)-b-PNIPAM (PAPMA-b-PNIPAM)169 and poly(2-(dimethylamino)ethyl methacrylate)-b-PNIPAM (PDMAEMA-b-PNIPAM). 170 Cross-linked polymersomes were obtained either by adding an oppositely charged polyelectrolyte complex with a PAPMA block or gold nanoparticles embedded in the PDMAEMA domain. After cross-linking, the vesicles were “locked” in place and could dissociate only upon swelling when the temperature was lowered. Similarly, Ding and co-workers prepared temper-

isopropylacrylamide) (PNIPAM) are the most widely reported because of particular interest in PNIPAM’s lower critical solution temperature (LCST) of 32 °C, which is just below the physiological body temperature. Thermally triggered assembly/ disassembly of these polymers can be exploited for drug delivery or injectable gelation. One of the first examples of this process used the diblock copolymer PEO-b-PNIPAM.166 This polymer is amphiphilic in water above body temperature (37 °C) and can self-assemble into vesicles, encapsulating both hydrophilic drugs in the aqueous lumen and hydrophobic molecules in the membrane. With a decrease in temperature, the PNIPAM block becomes hydrophilic, and the vesicles disassemble and release the encapsulated cargo. The PNIPAM block has also been conjugated to hydrophobic polymer blocks, and the resultant copolymers can form assemblies at room temperature that can further aggregate into complex morphologies at high temperatures. Moughton and O’Reilly167 reported a diblock copolymer (PtBuA-b-PNIPAM) in which the PNIPAM block had a permanently hydrophilic charged quaternary amine “headgroup”. They observed a thermally induced morphology transition from micelles to vesicles due to this unique structure. Mann and co-workers conjugated 666


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been studied for triggering the disruption and disassembly of AZO-containing polymersomes in a reversible manner.186−188 Zhang and co-workers synthesized a block copolymer composed of poly(acrylic acid) as the hydrophilic block and AZO-containing polyacrylate as the hydrophobic block, which self-assembled into giant spherical microvesicles in a mixture of water and THF.189 Upon irradiation with 365 nm light, the azobenzene side groups underwent trans-to-cis isomerization that induced a deformation of the vesicles from a spherical shape to an earlike shape. In another system, they prepared photoresponsive giant vesicles from block copolymers composed of poly(N-isopropylacrylamide) and AZO-containing polyacrylate and observed fusion of the vesicles upon irradiation with UV light.190 Yu and co-workers reported lightresponsive polymer vesicles formed from the self-assembly of a block copolymer consisting of hydrophilic PEG and hydrophobic azopyridine-containing polymethacrylate (PAP). The vesicles underwent photoinduced deformation cycles, including fusion, disintegration, and rearrangement, in H2O/THF mixtures upon exposure to UV light. In addition, this deformation cycle stopped with cessation of UV light exposure.186 Most of the aforementioned photoresponsive polymersomes are based on linear block copolymers. Other structural systems, such as host−guest interactions and dendritic block copolymers have also been explored. Ji and co-workers prepared the azobenzene-containing block copolymer poly(ethylene oxide)b-poly(6-(4-phenylazophenoxy)hexyl methacrylate-co-2(dimethylamino)ethyl methacrylate) (PEO-b-P(AzoMA-coDMAEMA)), which assembles into vesicles in water.191 Photostimuli control the inclusion and exclusion reactions of β-CD and azobenzene, therefore enabling reversible photoresponsive self-assembly and disassembly based on the wavelength of the irradiating light (365 or 450 nm). Huang and co-workers reported motif-photoresponsive polymersomes based on the molecular recognition between a water-soluble pillar[6]arene host and an azobenzene-containing amphiphilic guest.192 A reversible transition between vesicles and solid nanoparticles was achieved with the trans-to-cis photoisomerization of the AZO groups upon application of UV and visible light. Oriol and co-workers reported AZO-conjugated linear− dendritic block polymersomes composed of linear PEG segments linked to fourth generation 2,2-di(hydroxymethyl)propionic acid (bis-MPA)-based dendron-containing 4-isobutyloxyazobenzene units and hydrocarbon chains (C18) randomly connected to the periphery of the dendron.193−195 The selfassembly and light-responsiveness of the vesicles were explored. Both hydrophilic (rhodamine B) and hydrophobic (Nile red) compounds were encapsulated into the vesicles, and the phototriggered drug release and controlled polymer degradation were studied. Spiropyran (SP) is a well-known photochromic molecule that can undergo reversible isomerization between the hydrophobic ring-closed spiropyran (SP) form and the hydrophilic ring-opened merocyanine (MC) form under UV and visible light irradiation in a wavelength-selective manner. The light-tunable SP-to-MC isomerization process has been exploited to reversibly regulate the permeability of polymersomes. Liu and co-workers prepared photochromic polymersomes self-assembled from amphiphilic PEO-b-PSPA block copolymers, where SPA is an SP-based monomer containing a unique carbamate linkage.196 The phototriggered isomerization between hydrophobic SP (λ2 > 450 nm irradiation) and

ature-responsive vesicles based on poly(2-cinnamoylethyl methacrylate)-b-poly(N-isopropylacrylamide) (PCEMA-b-PNIPAM) and subsequently photo-cross-linked the PCEMA shells.171,172 Dual or multiresponsive polymersomes, especially those combining sensitivity to temperature and reduction, temperature and pH, or temperature and light, have been often investigated. Zhong and co-workers prepared PEG-b-PAA-bPNIPAM polymersomes by heating polymer solutions to 40 °C and cross-linking the PAA segments with reduction-sensitive cystamine (Cys) via carbodiimide chemistry. These cross-linked polymersomes keep their structures in phosphate-buffered saline at 37 °C but rapidly dissociate into unimers in response to 10 mM dithiothreitol (DTT).102,173 pH-responsive tertiary amines containing polymers often have temperature-dependent dissociation constants (pKa) and therefore have been integrated into polymersomes to exploit this temperature sensitivity. Lecommandoux and co-workers prepared the double hydrophilic block copolymer poly(2(dimethylamino)ethyl methacrylate)-b-poly(glutamic acid) (PDMAEMA-b-PGA) and investigated its pH- and temperature-driven self-assembly behavior.65 They demonstrated that the process of self-assembly into polymersomes and micelles could be tuned as a function of pH and/or temperature. Armes and co-workers investigated the ability to control the aqueous self-assembly of PMPC-b-PDPA diblock copolymers by manipulating the solution temperature.174 Other tertiaryamine-based temperature-sensitive morphology transitions have also been investigated, including those involving poly(2vinylpyridine) (P2VP)175 and poly(N,N-dimethylacrylamide)b-polystyrene-b-poly(N-(4-vinylbenzyl)-N,N-diethylamine) (PDMA-b-PS-b-PVEA).176 Temperature-sensitive polymers, such as poly(N-vinylcaprolactam) (PVCL),165,177 poly(propylene oxide)-b-poly(L-lysine),178 poly(trimethylene carbonate)-b-poly(L-glutamic acid),179 poly(trans-N-(2-ethoxy-1,3dioxan-5-yl)acrylamide),55 and modified poly(aspartamide),180,181 have also been reported. Typical polymer structures or reactions used for temperature-responsive polymersomes are listed in Table 1F. Light-Responsive Polymersomes. Light as a trigger for controlling drug release has received great attention as a remote control of drug release in an on/off switching manner with high spatial and temporal precision.32,182 The release profiles of such systems can be regulated by adjusting the light wavelength, intensity, and exposure time. Furthermore, light-responsive systems are often easily manipulated and do not require additional triggers or sensitive moieties.183,184 For these reasons, light-responsive polymersomes have potential for ondemand drug delivery and noninvasive clinical therapy.185 The photoresponsive polymersomes are commonly obtained by incorporating appropriate photoresponsive moieties into block copolymers. The corresponding polymersomes must then be disrupted and dissociated on the basis of the following two principles: (1) photoinduced structural and/or property changes, including the hydrophobic−hydrophilic balance and reversible photo-cross-linking, and (2) photoinduced polymer degradation or cleavage of block junctions. The most widely reported photoresponsive moieties include azobenzene (AZO), spiropyran (SP), 2-diazo-1,2-naphthoquinone (DNQ), Onitrobenzyl (ONB), and coumarin derivatives (Table 1G). Photoinduced Structural and/or Property Changes. The AZO moiety can reversibly transform from the trans isomer to the cis isomer upon UV and visible-light irradiation, which has 667


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Figure 14. (A) Schematic representation of a doxorubicin/USPIO-loaded and fluorescein isothiocyanate (FITC)-grafted polymersome. (B) Internalization of USPIO-loaded polymersomes in HeLa cells as observed by confocal fluorescence microscopy (after 24 h of exposition; scale bar =10 μm). (C) Cumulative drug release from USPIO/doxorubicin-loaded polymersomes upon exposure (or not) to a high-frequency alternating magnetic field (indicated by the arrows) at 37 °C in phosphate buffer (50 mM, pH 7.4). (D) MTT assay of HeLa cells after 72 h of exposure to USPIO-loaded polymersomes at a final doxorubicin dose of 12 μg/mL. Reprinted with permission from ref 89. Copyright 2013 Elsevier.

of hydrophilic network channels within the vesicle membranes and bilayer permeabilization. Hydrophilic and hydrophobic molecule encapsulation and light-switchable enzymatic biocatalysis have also been explored. Most of the aforementioned light-responsive polymersomes are based on irradiation with UV and visible light, which limits their application to the regions of the body that can be directly illuminated. For deeper clinical applications, near-infrared (NIR)-light-sensitive self-assemblies are ideal and promising candidates because of advantages including deep tissue penetration, low scattering loss, and minimal harm to tissues. Some NIR-light-responsive polymersomes have been synthesized by introducing chromophores that can respond to long wavelengths or exploit two-photon technology. One of these appealing strategies uses rare-earth-doped upconverting nanoparticles (UCNPs) for NIR-triggered drug release and photoswitching.202 When irradiated with NIR light, the UCNPs can absorb NIR light for conversion to higher-energy photons in the UV or visible region, which has been widely investigated for NIR-responsive fluorescence imaging, drug delivery, and photodynamic therapy (PDT) by coupling to an organic photosensitizer.202,203 A related strategy uses NIR-absorbing plasmonic materials, such as metal nanoparticles or organic chromophores, to convert photon energy to heat, a process called the photothermal effect, to trigger the release of chemotherapeutic molecules from NIR-light-responsive polymersomes.204−206 Nie and co-workers prepared gold nanoparticles and photosensitizer Ce6-loaded vesicles.205,207 The loaded gold nanoparticles produced heat under 671 nm laser irradiation, and the heating dissociated the vesicles, leading to the release of the Ce6 substrate to produce singlet oxygen for cancer therapy. The NIR-light-responsive polymersomes were studied in image-guided synergistic photothermal and photodynamic therapy of tumors in vivo. Magnetic-Field-Responsive Polymersomes. Magneticfield-responsive systems have promising biomedical applications in therapeutics, imaging, and diagnostics because of their

zwitterionic MC (λ1 < 420 nm irradiation) states endows the polymersome with photoswitchable and reversible bilayer permeability as well as switchable drug release triggered by alternating exposure to UV and visible light. Photoinduced Polymer Degradation or Cleavage. ONitrobenzyl derivatives have been investigated as photocleavable photochromic molecules. These photocleavable molecules are positioned in the main chain, side chain, or block junction of block copolymers, which breaks the hydrophobic−hydrophilic balance of the system, inducing the degradation of the backbone into oligomers under irradiation. Meier’s group synthesized an amphiphilic block copolymer using a photodegradable ONB linker as the junction point between hydrophilic PAA and hydrophobic ONB-substituted poly(γ-methyl-ε-caprolactone) (PMCL-ONB) blocks.197,198 The resultant vesicles disintegrated upon UV irradiation, yielding small micellar-like structures and simultaneously releasing the preloaded low-molecular-weight dye and proteins. Furthermore, the payload was released in a controlled manner by varying the UV intensity. Burdick and co-workers prepared photocleavable polymersomes with 2-nitrophenylalanine (2NPA) conjugating the PEG blocks and the hydrophobic (PCL) blocks.199 Liu et al.200 reported self-immolative polymersomes self-assembled from amphiphilic block copolymers consisting of a triggered degradable poly(benzyl carbamate) (PBC) block and a hydrophilic poly(N,Ndimethylacrylamide) (PDMA) block. The polymersomes could undergo head-to-tail cascade depolymerizaiton upon various triggers, including visible light, UV light, or a reductive milieu. In contrast to most reported vesicle dissociations or vesicle-to-unimer transitions upon irradiation, Liu and coworkers also prepared light-responsive polymersomes with stimuli-regulated traceless cross-linking.201 They synthesized block copolymers composed of PEG and 2-aminoethyl methacrylate functionalized with ONB in the side chain. During self-assembly into polymersomes, UV-triggered selfimmolative decaging releases primary amine moieties, leading to vesicle cross-linking, which is accompanied by the generation 668


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Figure 15. Structures of PS-β-CD and PEO-Fc and schematic illustration of the voltage-responsive controlled assembly and disassembly of PS-βCD/PEO-Fc supramolecular vesicles. Reprinted from ref 222. Copyright 2010 American Chemical Society.

noninvasive nature and ease of control.89,208,209 They are commonly synthesized by incorporating ferromagnetic or paramagnetic materials into the self-assemblies and have been widely investigated for magnetically triggered drug delivery systems and magnetic resonance imaging (MRI).210,211 Lecommandoux and co-workers reported polymersomes that self-assemble from the block copolymer poly(trimethylene carbonate)-b-poly(L-glutamic acid) (PTMC-b-PGA) and encapsulate both ultrasmall superparamagnetic iron oxide (γFe2O3) nanoparticles (USPIO NPs) and the anticancer drug Dox within the membrane.89,212 His group studied the magnetic-field-triggered drug release and the deformation of the vesicle membranes caused by local hyperthermia under an applied magnetic field (Figure 14). In their other systems, amphiphilic polybutadiene-b-poly(glutamic acid) (PBD-bPGA) diblock copolymers were used to prepare magnetic micelles and vesicles.208 Förster et al. prepared polymersomes loaded with Fe3O4 nanoparticles in the bilayer membrane at the hydrophobic−hydrophilic interface, which bridges adjacent bilayers and forms oligo- and multilamellar vesicles.211 Gong’s group213 and Du’s group214−217 prepared multifunctional SPIO/Dox-loaded polymer vesicles to investigate their potential use in targeted cancer therapy and MRI. Park and co-workers reported the self-assembly of magnetic nanoparticles based on the amphiphilic block copolymer PAA-bPS and their controlled morphology with magnetic nanoparticles distributed in different regions of the assembly.210 Webb and co-workers prepared Fe3O4 nanoparticle−vesicle assemblies embedded within a hydrogel extravesicular matrix and demonstrated remote magnetic-triggered payload release.218 Most of the reported magnetic-field-responsive systems were constructed by encapsulation of magnetic nanoparticles into polymersomes. In contrast, van Hest and co-workers used diamagnetic structures assembled from amphiphilic block copolymers for magnetic manipulation. They prepared bowlshaped polymer stomatocytes by highly regulating the shape of PEG-b-PS amphiphilic block copolymers.219 They were able to reversibly change the size of the bowl opening because of the highly anisotropic magnetic susceptibility of the PEG-b-PS building blocks.209 They then studied the capture and release of various payloads using this controlled opening/closing mechanism under magnetic fields.220,221 Electric-Field-Responsive Polymersomes. Electrical stimuli can change the charge or polarity of constituting

polymers, enabling the construction of electric-field-responsive polymersomes. These morphological changes can alter the chemical composition of a structure and have favorable applications in biological systems. Yuan and co-workers synthesized two terminally decorated homopolymers, polystyrene-β-cyclodextrin (PS-β-CD) and poly(ethylene oxide)-ferrocene (PEO-Fc), and prepared supramolecular vesicles based on the host−guest interactions between β-CD and Fc.222 The assembly/disassembly behavior of the vesicles was reportedly reversible on the basis of voltage through the reversible association/dissociation of this supramolecular connection (Figure 15). Park and co-workers reported an electric-potential-responsive vesicular system using the redox responsiveness of an amphiphilic rod−coil molecule, tetraaniline poly(ethylene glycol) (TAPEG).223 In aqueous solutions, the amphiphile TAPEG in its reduced leucoemeraldine base (LEB) form self-assembles into unilamellar vesicles, and upon application of an oxidizing voltage the vesicle membrane splits into smaller micellar objects, which then reassemble to form vesicles upon exposure to a reducing voltage. These electrically switchable vesicles were also exploited for molecular delivery. Ultrasound-Responsive Polymersomes. Ultrasound has been used as a promising stimulus because of its ease of administration, low cost, and deep penetration into the body.146,217 A variety of ultrasound-triggered release systems, including microemulsions,224 polymer micelles, 225 liposomes,226 and multilayered capsules,227 have been reported. Feijen and co-workers synthesized air-encapsulated PEG-bPLA polymersomes, and under a medical ultrasound, polymersome bubbles were visualized as bright spots using a medical ultrasound scanner.228 These air-containing polymersomes have potential applications in targeted ultrasound imaging and triggered drug release. Chen and Du217 reported ultrasound- and pH-sensitive dual-responsive polymersomes based on the block copolymer poly(ethylene oxide)-b-poly(2(diethylamino)ethyl methacrylate-stat-2(tetrahydrofuranyloxy)ethyl methacrylate) (PEO-b-P(DEAstat-TMA)). The effects of solution pH and duration of ultrasound radiation on the size and morphology of the assemblies were investigated. Controlled release of a loaded anticancer drug was achieved by altering the ultrasound exposure and pH of the solution. 669


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CONCLUSIONS AND FUTURE PERSPECTIVES The introduction of stimuli-triggered responsiveness allows polymersomes to recognize variations of physical, chemical, or biological conditions and activate corresponding changes in their intrinsic properties. Numerous biological and external stimuli, including pH, redox, enzymes, temperature, light, magnetic fields, electric fields, and ultrasound, have been employed to disrupt the hydrophobic−hydrophilic balance of polymersomes to destabilize their assemblies. Developments in polymer synthesis have made it possible to design polymersomes with tailorable physicochemical, pharmacological, and biological properties.27,229,230 Cross-linking or targeting moieties allow for enhanced in vitro and in vivo stability, prolonged circulation time, and improved accumulation at the target sites.19,27,231−233 Tunable membrane permeability and incorporation of specific channel-forming proteins have been investigated for cell mimics. The compartmentalization of polymersomes enables the study of cellular metabolism such as biochemical synthesis and enzymatic reactions. However, there are many scientific and engineering issues that must be addressed for translation. First of all, the biosafety of polymersomes should be considered primarily. Many of the reported polymersomes were evaluated in vitro and small animals and showed little toxicity; however, long-term toxicity and immunogenicity should be a concern,10 especially when these applications require systemic administration. To facilitate their translation to human studies, it is necessary to consider the potential biocompatibility of each building block as well as the degraded composition(s) of material utilized in their early design and development. Furthermore, most stimuli-responsive studies have been demonstrated under relatively static conditions with a limited number variables compared with a real situation. The promising results in vitro do not guarantee their efficacy in vivo.234 For example, in the design of delivery vehicles that can target the tumor site and respond to the physiological signals in the tumor microenvironment, a complex scenario of requirements must be considered, including long blood circulation time, ability to reach the target site, triggered release of cargo, and biodegradability of the carrier. Among those design clues, the precise integration of the responsiveness of a certain formulation and the targeted physiological signal (e.g., pH, enzyme activity, or ROS) remains challenging. To optimize the treatment efficacy, both the dynamic responsive behavior of the formulation and detailed information on the targeted physiological signal, such as the distribution and concentration at the diseased site, should be thoroughly investigated. On the other hand, external stimuli such as light, magnetic field, temperature, and electric field can be applied to tune the spatiotemporal action profile of a certain formulation and further enhance the performance.235 Last but not least, in regard to scalable production and reproducible manufacturing, significant efforts should be made in the design, synthesis, and optimization of amphiphilic block copolymers and the subsequent assembly procedures. A high-performance system with a simple and reliable fabrication process is always desired for translation.

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Zhigang Xie: 0000-0003-2974-1825 Zhen Gu: 0000-0003-2947-4456 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the American Diabetes Association (1-15-ACE-21), JDRF (3-SRA-2015-117-Q-R), and the NC TraCS, NIH’s Clinical and Translational Science Awards (CTSA) at UNC-CH 1UL1TR001111) to Z.G.

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