Stimuli-responsive polymersomes for drug delivery applications

Stimuli-responsive polymersomes for drug delivery applications

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Mónica Cristina García Unidad de Investigación y Desarrollo en Tecnología Farmacéutica (UNITEFA)-CONICETUNC, Departamento de Ciencias Farmacéuticas, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba, Argentina

13.1 Introduction In recent years, the potential use of nanotechnology in biomedical and pharmaceutical areas drove considerable attention, particularly the development of biocompatible nanostructured systems of therapeutically active agents, such as antitumor drugs, peptides and proteins, vaccines, and biotechnology-based therapeutics. Many nanomaterials have been employed as delivery vehicles for drugs and/or imaging agents to improve their efficacy, safety, solubility, permeability, bioavailability, and stability against physical, chemical, and enzymatic degradation [1]. Among all the types of drug nanocarriers that have been studied, several progresses in polymer nanoscience and nanotechnology have brought a revolutionary change to the development of new strategies for biomedical applications [2]. Polymeric carriers have considerable potential as drug delivery system [3] and, in the past decades, they have emerged as a very promising technology for gene therapy, bioimaging, biosensing, in vivo diagnosis, tissue engineering, and regenerative medicine [2, 3]. Different polymeric nanoparticles with distinctive sizes, shapes, and physicochemical and biopharmaceutical properties can be tailored for delivery of diverse drugs and imaging [4]. Some of these nanostructures include nanospheres, nanocapsules, nanogels, polymeric micelles, polymersomes, dendrimers, etc. [1]. In particular, 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 [5]. Polymersomes are artificial vesicles that have received significant attention due to their entangled architecture. They have spherical forms in which a hydrophilic core, that can encapsulate water-soluble molecules, is enclosed by a hydrophobic membrane, which may incorporate hydrophobic molecules [6] (Fig. 13.1). The membrane thickness of polymersomes can be tailored by shifting the hydrophobic ratio of the amphiphilic copolymers. Because of their macromolecular nature, the polymeric chains entangle between each other allowing an extra interaction between the copolymers. In consequence, their stability and toughness are superior that to their natural lipid counterpart, liposomes [6, 7]. Polymersomes have higher chemical and physical stability than liposomes. This is largely due to the higher molecular weight of copolymers compared with lipids, Stimuli Responsive Polymeric Nanocarriers for Drug Delivery Applications. https://doi.org/10.1016/B978-0-08-101995-5.00019-2 Copyright © 2019 Elsevier Ltd. All rights reserved.

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(A)

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Diblock copolymer Triblock copolymer Hydrophobic drug Hydrophilic drug

Fig. 13.1  Schematic illustrations of polymersomes based on (A) diblock and (B) triblock copolymers as carriers of hydrophilic and hydrophobic drugs.

which differs by at least one order of magnitude [1]. The molecular weights of lipids are generally <1 kDa, while those of block copolymers can be up to 100 kDa [5]. Moreover, polymersomes are chemically versatile, due to their physicochemical properties (such as: vesicle size, membrane thickness, biodegradation, permeability, stimuli-responsiveness, and among others) can be modified by selecting polymer chemical composition, appropriate molecular weight values and proportion between hydrophilic and hydrophobic blocks [8, 9]. Nevertheless, polymersomes usually show low encapsulation efficiency, which is limited to the concentration of the ­solution [10]. When discussing self-assembled nanostructures, there is a very important parameter to take into consideration known as hydrophilic volume fraction (f), which is commonly employed for amphiphilic block copolymers. This value is defined as the relation between the hydrophilic portion of the polymeric chain and the total molecular mass. For copolymers with polyethylene glycol (PEG) as hydrophilic chain and considering the density of homopolymers, it is possible to predict the type of nanostructure aggregation considering the f value [1]. In this sense, the main pathways for self-assembly of block copolymers into polymersomes have based on it. Vesicular nanostructures or polymersomes are preferentially formed at 25% < f < 40% [11, 12]. However, only this parameter does not guarantee the success of self-assembly of copolymers into polymersomes and the method chosen to develop them is a key factor for their formation [13]. After the self-assembly process, hydrophilic blocks on the outside of the vesicle adopt a brush-like configuration, which increases their colloidal stability and

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c­ amouflage ability against protein fouling [9]. Because of their colloidal stability and tunable and resistant membrane properties, polymersomes have been proposed as platforms for drug delivery, due to their ability to encapsulate a broad range of hydrophilic and hydrophobic agents, such as anticancer drugs, proteins, and genes [7, 9, 14]. More recently, applications include imaging and theranostics [15, 16]. Regarding the use of polymersomes as imaging platforms, they provide higher resolution than conventional techniques and allow in vivo monitoring of biological pathways and cellular functions, besides being noninvasive [9, 17]. Polymersomes can be obtained with different and very distinct sizes. By electroformation, lithography, microfluidic platform, and double emulsion micrometer polymersomes can be developed [9]. For obtaining nanometer polymersomes, the useful methods are film and bulk rehydration and solvent switch. In general, these methods need postpreparation process to achieve appropriate size distribution of the polymeric vesicles, which includes sonication, extrusion through a polycarbonate membrane with defined pores and freeze-thaw cycles [6, 9, 18, 19]. Most conventional methods commonly used for obtaining polymersomes have been adapted from liposome preparation techniques. The film rehydration is one of the most applied methods for lab-scale production. This method consists in a block copolymer solution in an organic solvent which is evaporated to a thin polymer film on a round-­bottomed flask. The rehydration of the polymer film is achieved by subsequently adding an isotonic aqueous medium, which leads to a detachment of the film from the glass surface. The rehydration and swelling process can be influenced by stirring, shaking, or sonication, which affects to some extent the resulting polymeric vesicle size. In general, this method allows obtaining unilamellar and multilamellar vesicles with a rather broad size distribution [6, 20]. Some amphiphilic block copolymers allow a direct dissolution from bulk material. In that cases, longer and even more vigorous agitation is required for the complete rehydration of the polymer [21]. In the cosolvent method, also called solvent displacement or nanoprecipitation method, the amphiphilic polymer is dissolved in a water-miscible solvent, after that is added dropwise into water under vigorous stirring, and then the water-miscible solvent is subsequently removed by dialysis, freeze-drying, or evaporation [6]. It is important to note that the most methods for polymersomes preparation still have low reproducibility and feasibility for up-scaling, which stays a big issue with regard to the translation into clinical application [6]. Current studies have focused on the development of polymersomes with different shapes (tubular, toroidal, and higher genus particles). As well, recent variation refers to patchy polymersomes, prepared by mixing different types of copolymers [1]. Moreover, the properties of polymersomes can be broadly tailored and modified taking advantage of the high flexibility and customizability of block copolymers. A wide range of polymersomes with diverse sizes, architectures, surface properties, membrane properties such as permeability, and chemical functionalities has been reported up to date [5]. Another rising research field is polymersomes as compartments for in situ reactions at the nanoscale (nanoreactors) [8, 17]. The cutting-edge and emerging research focuses involve the design and development of stimuli-responsive polymersomes, also called “smart” polymersomes, that can

Stimuli Responsive Polymeric Nanocarriers for Drug Delivery Applications

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N a n o r e a ctor Fig. 13.2  Schematic representation of various stimuli-responsive polymersomes for different biomedical applications. Reproduced with permission from X. Hu, Y. Zhang, Z. Xie, X. Jing, A. Bellotti, Z. Gu, Stimuli-responsive polymersomes for biomedical applications, Biomacromolecules 18(3) (2017) 649–673. Copyright 2017, American Chemical Society.

recognize some environmental stimuli and release the drug loaded in dose-, s­ patial-, and temporal-controlled manners. Internal biological-stimuli include endolysosomal pH, redox potential, enzymatic activities, and monosaccharide concentration. External physical stimuli include temperature, light, electric field, mechanical force, and ultrasound (Fig. 13.2). Responsiveness to these types of stimuli can be exploited in a drug delivery system by incorporating intrinsically stimuli-responsive chemical groups into the amphiphilic block copolymer. The obtained polymersomes are then capable to undergo physical and chemical changes (e.g., swelling, membrane fusion, disassembly, and bond cleavage) in response to specific stimuli, thus subsequently leading to polymersome disruption and drug release [5, 7, 9]. In this perspective, this chapter aims to cover the recent advances in stimuli-­ responsive polymersomes, including biological-stimuli such as pH-responsive, redox-­ responsive, enzyme-responsive, glucose-responsive, and gas-responsive, as well as external-stimuli such as temperature-responsive, light-responsive, magnetic field-­ responsive, and ultrasound-responsive. Aspects related to the mechanism involved in the drug release and some examples of their main biomedical applications will be highlighted. Also, the use of stimuli-responsive polymersomes for cancer therapy will be stressed.

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13.2 Biological-stimuli-responsive polymersomes Polymersomes loaded with any drug can respond to internal stimuli to release the drug. Changes in the internal microenvironmental such as pH, redox potential, enzyme, glucose, and gas could produce a conformational change in the nanostructure and the drug loaded can be immediately released. In the following sections, general aspects and some examples of biological-stimuli-responsive polymersomes are detailed.

13.2.1 pH-responsive polymersomes pH-responsive polymersomes are among the most studied stimuli-responsive nanostructures because of the presence of physiological pH gradients within the body. The extracellular pH of tumor and inflammatory tissues (~6.5–7.2) is slightly lower than that of normal tissues and other biological fluids (~7.4); it is further decreased in intracellular endosomes (pH 5.5–5.0) and lysosomes (pH 4.0–4.5). Owing to the wide range of pH gradients in physiologic systems, the application of pH-responsive polymersomes as drug delivery carriers has been widely exploited to deliver drugs to target locations, including intracellular compartments, specific organs, or microenvironments associated with certain pathological situations [5, 7]. These types of polymersomes are generally constructed by incorporating acid-cleavable bonds or ionizable groups into the block copolymer or by directly forming polyionic complexes via electrostatic interactions [22–26]. The most straightforward examples of pH-responsive polymersomes are based on hydrolysis-susceptible aliphatic polyesters, such as poly(lactic acid) (PLA) or poly(e-caprolactone) (PCL), which were used as the hydrophobic block. As shown in Fig. 13.3, Ahmed et al. revealed pH-triggered hydrolytic degradation of the block copolymers, as evidenced by the morphological transitions of the polymersomes to micelles which allow releasing the payload. In vivo experiments demonstrated that a single systemic injection of the dual drug combination showed a higher maximum tolerated dose than the free drug cocktail and shrinks tumors more effectively and more sustainably than free drug [14]. Dan and Ghosh have shown the synthesis of an amphiphilic triblock copolymer by sequential thiol-acrylate Michael addition reaction in one pot. The copolymer segmented by acid-labile β-thiopropionate linker, which showed spontaneous vesicular assembly, and the stimuli-responsive disassembly at mild acidic conditions (pH 5.5), resulting in the sustained release of noncovalently encapsulated molecules [27]. It has been reported several acid-cleavable linkers, namely hydrazone, imine, ortho ester, and acetal for the preparation of polymersomes with tunable degradation kinetics. These types of linkers can be integrated into the main chain or the pendant chains of the block copolymer [5]. For example, Li et al. reported the useful of 2-[3-[5-amino-­1carboxypentyl]-ureido]-pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs). The synthetized polymersomes displayed highly efficient loading of both model proteins evaluated (bovine serum albumin and cytochrome C) and the in vitro release studies showed that protein release was markedly accelerated at mildly acidic pH due to the hydrolysis of acetal bonds in the vesicular membrane.

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Fig. 13.3  PEG–PLA-based polymersome self-assembly, degradation, and drug release. (A) Cryo-transmission electron microscopy (TEM) images of empty polymersomes. 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) Magnified views show key features of degradable polymersomes, including evidence of the vesicular lumen, the corona-lined pore, the thickening in membrane-micelle transition, and the exclusion zone that indicates an invisible layer of PEG around the micelles. (C) In vitro release and leakage of doxorubicin (Dox) and paclitaxel (TAX) from degradable and nondegradable polymersomes. Reproduced with permission from F. Ahmed, R.I. Pakunlu, A. Brannan, F. Bates, T. Minko, D.E. Discher, Biodegradable polymersomes loaded with both paclitaxel and doxorubicin permeate and shrink tumors, inducing apoptosis in proportion to accumulated drug, J. Contr. Release 116(2) (2006) 150–158, Copyright 2006.

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Fig. 13.4  Schematic illustration on PSMA-targeting pH-sensitive biodegradable chimaeric polymersomes for active loading and triggered intracellular release of GrB (apoptotic protein) into prostate cancer cells. Reproduced with permission from X. Li, W. Yang, Y. Zou, F. Meng, C. Deng, Z. Zhong, Efficacious delivery of protein drugs to prostate cancer cells by PSMA-targeted pH-responsive chimaeric polymersomes. J. Contr. Release 220 (2015) 704–714, Copyright 2015.

The prostate-specific membrane antigen (PSMA)-targeted polymersomes showed a long circulation time in nude mice and they showed promising properties as efficient protein nanocarriers for targeted prostate cancer therapy (Fig. 13.4) [28]. Liu’s group reported the preparation of pH-responsive polymersomes via supramolecular self-assembly of amphiphilic diblock copolymers, poly(ethylene oxide)-bpoly(2-((((5-methyl-2-(2,4,6-trimethoxyphenyl)-1,3-dioxan-5-yl)methoxy)carbonyl) amino)ethyl methacrylate) (PEO-b-PTTAMA), which were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization of a pH-responsive monomer using a PEO-based macroRAFT agent. The obtained amphiphilic diblock

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copolymer can self-assembled into vesicles consisting of hydrophilic PEO coronas and pH-responsive hydrophobic bilayers composed by cyclic benzylidene acetals, which were relatively stable under neutral pH, whereas they underwent hydrolysis upon exposure to acidic pH conditions. The release of the drugs loaded (Nile red and doxorubicin as hydrophobic and hydrophilic model drugs, respectively) was remarkably regulated by the solution pH values. The fabricated pH-responsive polymersomes were easily taken up by HeLa cells and were primarily located in the acidic organelles after internalization, where the pH-responsive cyclic acetal moieties were hydrolyzed and then the payloads were released, allowing for on-demand release of the molecules mediated by intracellular pH (Fig. 13.5) [22]. Yang and coworkers prepared pH-responsive polymersomes from co-assembly of amphiphilic cholate grafted poly(l-lysine) and acid-cleavable polymer−drug conjugate (PEG-doxorubicin conjugate) obtained via an acid-labile benzoic imine bond. This bond resulted in a pH-responsive membrane permeability and triggered dissociation of the polymersome following a drop in environmental pH [29]. On the other hand, nanostructures with ionizable groups can be also obtained. These pH-responsive polymersomes typically have weakly acidic groups such as carboxylic or sulfonic acids (i.e., polyacids) and/or weak basic groups such as primary, secondary, or tertiary amine groups (i.e., polybases) [5, 30]. They can suffer changes in conformation or solubility in response to changes in environmental pH via ionization (protonation or deprotonation) [5]. Liu and Eisenberg observed the pH-triggered morphological change of the triblock copolymer poly(acrylic acid)-b-polystyrene-b-poly(4-vinylpyridine) (PAA-b-PSb-P4VP) from vesicles to solid spherical aggregates and then back to vesicles. The segregation is based on the difference in repulsive interactions within the PAA or P4VP corona under different pH conditions. Vesicles with PAA on the outside can be inverted to P4VP on the outside by changing the pH while the vesicles have swollen cores and are under dynamic conditions [31]. Chiu et  al. reported multivesicle assemblies with pH-responsive transmembrane channels in the vesicle walls made by two-step double emulsion of copolymers comprising acrylic acid (AAc) and acrylate of 1,2-distearoyl-rac-glycerol [distearin acrylate (DSA)]. They observed that pH of 5.0 the channels were closed because of hydrogen bonds and hydrophobic association of deionized AAc. At pH of 6.5 ionization of AAc occurred leading to disruption of the hydrogen bonds and hydrophobic association and the formation of permeable channels. This pH-responsive on/off process is reversible, and it relies on change in pH value in a narrow range between 6.4 and 6.5 (Fig. 13.6). The channels were accessible to both small and large molecules, such as calcein and hemoglobin, respectively [5, 32]. Du and Armes reported that the membrane permeability of pH-responsive polymersomes composed of the self-cross-linkable copolymer PEO-b-poly(2diethylamino ethyl methacrylate)-stat-3(trimethoxysilyl) propyl methacrylate (PEOb-P(DEAEMAstat-TMSPMA)) can be tuned by pH while preserving their shape. Cross-linking was accomplished by the reaction of the trimethoxysilyl groups after hydrolysis into a siloxane network. At low pH, protonated PDEAEMA blocks led the membrane to swell, along with increasing permeability. Also, they found that higher degrees of cross-linking resulted in lower wall permeability [33].

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Fig. 13.5  Schematic representation of pH-responsive polymersomes self-assembled from poly(ethylene oxide)-b-poly(2-((((5-methyl-2-(2,4,6-trimethoxyphenyl)-1,3-dioxan-5-yl) methoxy)carbonyl)amino)ethyl methacrylate) (PEO-b-PTTAMA) amphiphilic diblock copolymers containing acid-cleavable cyclic acetal side linkages in the hydrophobic block. Upon internalization by HeLa cells via endocytosis, the polymersomes are subjected to spontaneous hydrolysis, resulting in the disruption of vesicular nanocarriers and concomitant release of encapsulated drugs. Reproduced with permission from L. Wang, G. Liu, X. Wang, J. Hu, G. Zhang, S. Liu, Acid-disintegratable polymersomes of pH-responsive amphiphilic diblock copolymers for intracellular drug delivery, Macromolecules 48(19) (2015) 7262–7272, Copyright 2015 American Chemical Society.

Amphiphilic block copolymers composed of polypeptides are a versatile class of stimulus-responsive building blocks for the self-assembly into vesicles and they have attracted considerable interest for their high biocompatibility, biodegradability, and complex secondary conformations [5, 30]. In this sense, hybrid block copolymers where the polypeptide serves as hydrophobic and the synthetic polymer as the hydrophilic block have been used for the fabrication of this type of polymersomes. Also, block copolymers in which both the hydrophilic and hydrophobic blocks are ­polypeptides have

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been employed [34]. 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 used, which regulate the morphology of peptide-based nanoparticles. Moreover, the ionizable groups can also be used to interact with oppositely charged drugs or bioactive macromolecules via electrostatic interactions. Polymersomes formed from polypeptide-based copolymers, also known as pepsomes, have been developed and evaluated for several biomedical applications [5]. Cationic polypeptides such as poly(l-lysine) (PLys), poly-histidine (PHis), and ­poly-arginine (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, to form amphiphilic polypeptides. The research groups of Forster and Lecommandoux synthesized PGA- and PLys-based polypeptide block copolymers containing polybutadiene (PBD), polyisoprene (PI), or poly(trimethylene carbonate) (PTMC) [35–37]. They investigated the pH-responsive polymersomes that results from the polyelectrolyte corona and secondary structure of PGA or PLys in the block copolymers. Doxorubicin-loaded polymersomes exhibited high loading efficiency and high stability at room temperature, and the drug-release rate increased at acidic pH or with increasing temperature [38]. Rodriguez-Hernandez and Lecommandoux reported the formation of reversible polymersomes as a function of pH in water that can be produced in moderate acidic or basic aqueous solutions from zwitterionic polypeptide diblock copolymers (PGA-b-PLys) (Fig. 13.7). They anticipate that these pH-sensitive nanostructures are very promising candidates in macromolecular nanobiotechnologies and propose to study encapsulation strategies of different drugs and proteins and to evaluate the delivery properties as a function of pH [26]. In addition, Kataoka and coworkers fabricated some 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 [39]. The resultant polymersomes, also known as polyion complexes (PICsomes), exhibited several advantages compared with traditional polymersomes (they do not require organic solvent and are the encapsulation of water-soluble macromolecules is easer) [5]. Kataoka’s group prepared PICsomes with precise control of the size, distribution, and structure and obtained stable polymersomes with tunable membrane permeability by

Fig. 13.6, Cont’d  (A) Schematic representation of multivesicle assemblies equipped with pH-responsive transmembrane channels from two-stage double emulsion of poly(acrylic acid-co-distearin acrylate) (poly(AAc-co-DSA)). The AAc-rich regions and the bilayer islets within the vesicle membrane are not drawn to scale. (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). Reproduced with permission from X. Hu, Y. Zhang, Z. Xie, X. Jing, A. Bellotti, Z. Gu, Stimuli-responsive polymersomes for biomedical applications, Biomacromolecules 18(3) (2017) 649–673, Copyright 2017 American Chemical Society.

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Fig. 13.7  Schematic representation of the self-assembly of the diblock copolymer poly(glutamic acid)-b-poly(l-lysine) (PGA-b-PLys) into polymersomes. Reproduced with permission from J. Rodríguez-Hernández, S. Lecommandoux, Reversible inside-out micellization of pH-responsive and water-soluble vesicles based on polypeptide diblock copolymers, J. Am. Chem. Soc. 127(7) (2005) 2026–2027, Copyright 2017 American Chemical Society.

chemical cross-linking of the PIC layer. Also, they found that cross-linked PICsomes attained an extremely long-circulation property in the bloodstream [39]. As it can be seen, pH-responsiveness is one of the main stimuli applied in the design of responsive polymersomes and their biomedical applications. As it can be seen, pH-­ responsiveness is one of the main stimuli applied in the design of responsive polymersomes and their biomedical applications are of great interest in order to improve therapies.

13.2.2 Redox-responsive polymersomes Redox potentials are quite different in the intracellular and extracellular environment, and between tumor and normal tissue. In the extracellular environment, body fluids (e.g., blood), and on the cell surface, the concentration of one of the most prominent reducing agents, glutathione (GSH) is lower (2–20 μM) in comparison with cytosol and nuclei, where the concentration is much higher (10 mM), producing a great reductive microenvironment. Therefore, taking advantage of the local redox state and the large difference in redox potential (100–1000 times) between extra- and intracellular compartments, reduction or oxidation of responsive nanostructures can be used to change the properties of the polymersome membranes or to endow these compartments with novel functions, including triggered intracellular delivery of a variety of biologically active molecules from polymeric vesicles inside the cells [30, 40, 41]. Disulfide bonds are known to be responsive to reduction. They can be reduced to two thiols in the presence of reducing agents such as GSH. Disulfide bonds are simply introduced in the middle or side chain of an amphiphilic polymer. The dissociation energy of a disulfide bond (SS) is lower than that of a carbon-carbon bond. This type of bond is stable in an oxidizing extracellular environment, under physiological conditions; however, they are readily reduced in an intracellular environment due to the

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thiol-disulfide exchange reaction with GSH. Thus, cleavage of these bonds can induce disassembly of the polymersomes in reductive environments [5, 7, 30]. Moreover, the incorporation of disulfide bonds as cross-linkers in the nanostructure allows improving the stability of polymersomes while maintaining their responsiveness [5]. Even though several reduction-responsive amphiphilic copolymers have been reported, the number of studies involving reduction-responsive polymersomes is much lower. Xu et  al. reported water-soluble temperature-responsive triblock copolymers based on poly(ethylene oxide)-b-poly(acrylic acid)-b-poly(N-­isopropylacrylamide) (PEO-PAA-PNIPAM), which were prepared in one pot by RAFT polymerization using a PEO-trithiocarbonate (PEO-S-1-dodecyl-S-(R,R-dimethyl-R-aceticacid) trithiocarbonate) as a macro-chain transfer agent. From them, cross-linked ­ ­polymersomes were obtained, and they showed remarkable stability against dilution, organic solvent, high salt conditions and change of temperature in water; however, the polymersomes could rapidly dissociate under reductive conditions mimicking intracellular environment, quickly releasing the preloaded sample protein, FITC-dextran [42]. Zhong’s group also reported reduction and pH dual-responsive cross-linked polymersomes based on the poly(ethylene glycol)-poly(acrylic acid)-poly(2-(diethyl amino)ethyl methacrylate) (PEG-PAA-PDEAEMA) triblock copolymer, which were synthesized by controlled RATF polymerization and further modified with cysteamine to yield the thiol-containing PEG-PAA(SH)-PDEAEMA copolymer. This copolymer formed robust and monodisperse polymersomes with an excellent colloidal stability, which were rapidly dissociated in response to 10 mM glutathione at neutral or mildly acidic conditions (Fig. 13.8). These polymersomes showed potential for efficient intracellular delivery of proteins and potent induction of cancer cell apoptosis [43]. This group also reported reversibly stabilized multifunctional dextran nanostructures based on dextran-lipoic acid derivatives (Dex-LAs), in which the lipoic acid (LA) is responsible for the cross-linking and the reduction response. They were obtained for the triggered intracellular delivery of doxorubicin [44]. Zou et al. reported multifunctional polymersomes based on co-self-assembled from poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate) (PEG-P(TMC-DTC)) and cNGQGEQc-functionalized PEG-P(TMC-DTC) (cNGQ-PEG-P(TMC-DTC)). The membrane-forming hydrophobic block consists of P(TMC) backbone, and pendant dithiolane ring, which is analogous to LA. The cyclic peptide cNGQ was decorated on the surface. The obtained doxorubicin-loaded, cNGQ-decorated polymersomes showed efficient loading and targeted delivery of doxorubicin to subcutaneous as well as orthotopic A549 human lung cancer xenografts in nude mice (Fig. 13.9) [45]. Li and coworkers reported developed a robust reduction-responsive polymersome based on the amphiphilic block copolymer PEG-SS-polyacrylate/cholesterol (PAChol). The reduction sensitivity was introduced by the disulfide bridge that links the hydrophilic PEG block and the hydrophobic PAChol block. The obtained polymersomes showed physical stability for the presence of cholesterol as cross-linker. They observed that calcein release from PEG-SS-PAChol polymersomes was triggered by GSH [46]. On the other hand, oxidation-responsive polymersomes can be also obtained. Reactive oxygen species (ROS)-responsive nanostructures are an emerging class of biomaterial in the field of internal biological-stimuli. ROS mostly consists of h­ ydrogen

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Fig. 13.8  Schematic representation of reduction- and pH-bioresponsive disulfide-cross-linked polymersomes based on poly(ethylene glycol)-poly(acrylic acid)-poly(2-(diethyl amino)ethyl methacrylate) (PEG-PAA-PDEAEMA) triblock copolymer further modified with cysteamine to yield the thiol-containing PEG-PAA(SH)-PDEAEMA copolymer for efficient loading and triggered intracellular release of proteins. Reproduced with permission from H. Sun, F. Meng, R. Cheng, C. Deng, Z. Zhong, Reduction and pH dual-bioresponsive crosslinked polymersomes for efficient intracellular delivery of proteins and potent induction of cancer cell apoptosis, Acta Biomater. 10(5) (2014) 2159– 2168. Copyright 2014.

peroxide (H2O2), peroxynitrite (ONOO−), hydroxyl radical (OH), and superoxide (O2 -), which are produced from various endogenous sources and serve crucial roles in ­physiological processes, namely cellular signaling and proliferation, apoptosis, and immune responses. However, overproduction of ROS may result from oxidative stress, a biological feature that is accompanied by various pathological disorders such as cancer, infections, inflammation, cardiovascular disease, and diabetes. Taking advantage of the abnormal redox states in tumor and inflammatory tissues and considering that pathological sites possess distinctive characteristics from their surroundings, these sites have been considered as targets for site-specific delivery of therapeutic and imaging agents [5]. There are limited examples of oxidation-responsive polymersomes. Hubbell and coworkers reported the first ones, using oxidative conversion to destabilize polymersomes. They synthesized the triblock copolymer poly(ethylene glycol)-b-poly(propylene sulfide)-b-poly(ethylene glycol) (PEG-b-PPS-b-PEG) that self-assemble into unilamellar vesicles in aqueous solutions. The hydrophobic blocks based on PPS can suffer oxidative conversion from a hydrophobe to a hydrophile, poly(propylene sulfoxide) and ultimately poly(propylene sulfone). This oxidation process produces morphological changes from stable vesicles to wormlike micelles to spherical micelles

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Fig. 19.9  Figure legend continued on next page.

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and ultimately to nonassociating unimolecular micelles [47]. Also, Hubbell’s group evaluated the oxidation-sensitive nanoscale polymersomes based on the block copolymer PEG-b-PPS and applied them for both antigen and adjuvant delivery to dendritic cell endosomes. They observed that the polymersomes can function as a vaccine delivery platform for inducing cell-mediated antigen-specific immune responses [48]. Moreover, boronic esters have been widely investigated as oxidation-sensitive materials for H2O2-induced degradation. Deng et  al. reported oxidation-responsive multifunctional polymersomes, which exhibited intracellular milieu-triggered vesicle bilayer cross-linking, permeability switching, and enhanced imaging/drug-release features. Mitochondria-targeted H2O2 reactive polymersomes were obtained through the self-assembly of amphiphilic block copolymers containing arylboronate ester-capped self-immolative side linkages in the hydrophobic block, followed by surface functionalization with targeting peptides (Fig. 13.10). The co-incubation with H2O2 under simulated biological conditions (~1 mM H2O2) or under cellular oxidative milieu inside live cells led to cleavage of arylboronate capping moieties at first, followed by cascade decaging reactions and generation of primary amine moieties. The latter further resulted in vesicle cross-linking and concomitant permeabilization of bilayer membranes [49]. Gu’s group reported vesicles that quickly dissociate and release encapsulated insulin under the local hypoxic environment, caused by the enzymatic oxidation of glucose in the hyperglycemic state [50]. Also, this group reported hypoxia/H2O2 dual-sensitive vesicles obtained by conjugating 6-(2-nitroimidazole) hexylamine to hyaluronic acid and conjugating (2-nitroimidazol-1yl)methanethiol to the block copolymer PEG-bpolyserine, respectively. The polymersomes can disassociate and subsequently release insulin triggered by H2O2 and hypoxia generated during glucose oxidation catalyzed by glucose-specific enzyme. Under hypoxic conditions, the hydrophobic 2-­nitroimidazol groups were converted to hydrophilic 2-aminoimidazole groups catalyzed by nitroreductases, and meanwhile, the thioether linker served as a H2O2-sensitive moiety and was converted into a more hydrophilic sulfone by H2O2 (Fig. 13.11) [51]. Xu’s group has widely studied the use of selenium/tellurium-containing polymers. They are active electron donors and their reactions are potentially reversible, making them suitable to mimic the natural redox-involved metabolism [52, 53]. Fig. 13.9, Cont’d  Schematic representation of multifunctional polymersomes based on co-self-assembled from poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate) (PEG-P(TMC-DTC)) and cNGQGEQc-functionalized PEG-P(TMCDTC) (cNGQ-PEG-P(TMC-DTC)) for efficient loading and targeted delivery of doxorubicin to subcutaneous as well as orthotopic A549 human lung cancer xenografts in nude mice. The obtained polymersomes while sharing basic characteristics of pegylated liposomal doxorubicin (Lipo-Dox) including a vascular structure, small size, high drug loading and long circulation time presents several unique features such as easy synthesis, inhibited premature drug release, high tumor selectivity and accumulation, efficient intracellular drug release, low systemic toxicity, and high therapeutic index. Reproduced with permission from Y. Zou, F. Meng, C. Deng, Z. Zhong, Robust, tumorhoming and redox-sensitive polymersomal doxorubicin: a superior alternative to Doxil and Caelyx? J. Contr. Release 239 (2016) 149–158, Copyright 2016.

Stimuli-responsive polymersomes for drug delivery applications361 O O

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Fig. 13.10  Schematic representation of the preparation of oxidation-responsive multifunctional polymersomes exhibiting intracellular milieu-triggered vesicle bilayer crosslinking, permeability switching, and enhanced imaging/drug-release feature. Mitochondriatargeted reactive polymersomes were obtained through the self-assembly of amphiphilic block copolymers containing arylboronate ester-capped self-immolative side linkages in the hydrophobic block, followed by surface functionalization with targeting peptides. Upon cellular uptake, mitochondrial H2O2 triggers cascade decaging reactions and releases primary amine moieties; a prominent amidation reaction then occurs due to suppressed amine pKa within hydrophobic membranes, resulting in concurrent cross-linking and hydrophobic-tohydrophilic transition of polymersome bilayers inside live cells. Reproduced with permission from Z. Deng, Y. Qian, Y. Yu, G. Liu, J. Hu, G. Zhang, et al., Engineering intracellular delivery nanocarriers and nanoreactors from oxidation-responsive polymersomes via synchronized bilayer cross-linking and permeabilizing inside live cells, J. Am. Chem. Soc. 138(33) (2016) 10452–10466, Copyright 2016 American Chemical Society.

13.2.3 Enzyme-responsive polymersomes Enzyme-responsive polymersomes can offer unique properties, as a result of the high level of sensitivity, selectivity, and efficiency accompanied by enzymatic conversions for targeted delivery of therapeutic agents at specific sites. Specific enzymatic reactions have been utilized to develop smart materials that are switched between assembled and disassembled structures [5, 30]. In general, enzymes play crucial roles in several biological and metabolic processes, serving as the prime protagonists in the

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Fig. 13.11  Schematic representation hypoxia/H2O2 dual-sensitive polymersomes of the delivery of insulin (glucose-responsive polymersome-based vesicles, d-GRPs). (A) Formation and mechanism of d-GRPs comprised of poly(ethylene glycol)-polyserine modified with 2-nitroimidazole via a thioether moiety (PEG-poly(Ser-S-NI)). (B) Schematic of local inflammation induced by non-H2O2-senstive GRP-loaded patch, and schematic of d-GRPloaded patch for in vivo insulin delivery triggered by a hyperglycemic state for potential prevention of the long-term side effect associated with inflammation. Reproduced with permission from J. Yu, C. Qian, Y. Zhang, Z. Cui, Y. Zhu, Q. Shen, et al., Hypoxia and H2O2 dual-sensitive vesicles for enhanced glucose-responsive insulin delivery, Nano Lett. 17(2) (2017) 733–739, Copyright 2017 American Chemical Society.

chemistry of living organisms. The upregulation of enzymes often occurs in many diseases including cancer, thrombosis, inflammation, and infections [7]. Enzymeresponsive polymersomes are ideal candidates for drug delivery vehicles because both hydrophilic and hydrophobic drugs can be encapsulated [30]. Pramod et al. prepared dextran vesicular nano-scaffolds based on polysaccharide and a renewable-resource alkyl tail, which were used for dual encapsulation of water-­ soluble molecules like Rhodamine-B and the polyaromatic anticancer drug camptothecin, which were selectively loaded in the hydrophilic lumen and hydrophobic

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A Versatile Dual Encapsulation Polymer Carrier for Hydrophilic & hydrophobic Drugs Core encapsulation ble olu s ter est Wa gu

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Fig. 13.12  Dextran vesicular approach for delivery of hydrophilic and hydrophobic drugs (or molecules) into cells. Reproduced with permission from P. Pramod, K. Takamura, S. Chaphekar, N. Balasubramanian, M. Jayakannan, Dextran vesicular carriers for dual encapsulation of hydrophilic and hydrophobic molecules and delivery into cells, Biomacromolecules 13(11) (2012) 3627–3640, Copyright 2012 American Chemical Society.

membrane, respectively. The aliphatic ester linkage connecting the hydrophobic tail with dextran was demonstrated to be cleaved by esterase under physiological conditions for fast release of camptothecin or Rhodamine-B. The drug-loaded polymersomes demonstrated significantly better cellular uptake compared with free camptothecin and they were seen to localize in the perinuclear region of the cells (Fig. 13.12) [54, 55]. Jayakannan’s group also prepared pH- and enzyme-responsive polymersomes to deliver doxorubicin into breast cancer cells. Dextran was suitably modified with a renewable resource 3-pentadecyl phenol unit through imine and aliphatic ester chemical linkages that acted as pH and esterase enzyme stimuli, respectively [56]. Noncovalent host-guest interactions can also be introduced into enzyme-­responsive polymersomes. In this sense, Guo et  al. reported an enzyme-responsive vesicle with p-sulfonatocalix[4]-arene (SC4A) as the macrocyclic host and natural enzyme-­ cleavable myristoylcholine as the guest molecule. The self-assembled nanostructure exhibited highly specific and efficient responsiveness to cholinesterase, a key protein overexpressed in Alzheimer’s disease, endowing fast release behavior of entrapped water-­soluble drugs in presence of this enzyme [57]. In contrast to bond formation or cleavage in responsive materials, Deming’s group reported that o­ xidation/reduction reactions induced a hydrophobic/hydrophilic transition of the copolymer, resulting in vesicle disruption and payload release. They first prepared a fully ­hydrophobic ­precursor

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d­iblock copolypeptide, poly(l-methionine)-b-poly(l-leucine-stat-l-­phenylalanine), M65-b-(L0.5/F0.5)20. The direct oxidation in water of this precursor produced the amphiphilic Mo derivate (methionine sulfoxide), Mo65-b-(L0.5/F0.5)20. This derivate self-­ assembled into vesicles in water, which served as the substrate for methionine sulfoxide reductase enzymes. The hydrophilic Mo segments in the vesicles regenerated hydrophobic ­poly(l-methionine) segments (M) upon exposure to these enzymes, which resulted in a change in supramolecular morphology, from spherical to a crumpled sheet like, that caused vesicle disruption and release of the payload [58]. In addition, Habraken et  al. constructed biohybrid poly(l-glutamic acid-coalanine)-b-poly(n-butyl acrylate) (P(GA-co-Ala)-b-PBAc) and poly(l-glutamic acid-­ coalanine)-b-polystyrene (P(GA-co-Ala)-b-PS) block copolymers with various quantities of l-alanine by N-carboxyanhydride ring opening polymerization. When the self-­assembled nanostructures were exposed to the enzymes elastase and thermolysin, they were degraded depending on the polypeptide composition. The hybrid vesicles or micelles of the block copolymers possessed varying degrees of enzyme responsiveness when exposed to these enzymes, and enzymatic degradation of parts of the polypeptide block resulted in particle destabilization [59]. On the other hand, lysosomal enzymes like Cathepsin B are more abundant in tumor tissues than in normal tissues. These enzymes can be used to cleave certain ­peptide sequences such as Gly-Phe-Leu-Gly (GFLG). Polymersomes containing these peptide sequences can be transformed by the cleavage of the peptide, resulting in the release of a covalently bound drug, which make them good candidates for intracellular delivery. In this sense, Lee et  al. reported lysosomally cleavable polymersomes, in which a GFLG degradable linker was introduced to the middle of a hydrophilic methoxy poly(ethylene glycol) (mPEG) and a hydrophobic PLA block copolymer. The copolymer self-assembled into polymersomes in aqueous solutions and disassembled in the presence of Cath B at pH 5.5. Antiepidermal growth factor receptor-antibody was immobilized on the surface of polymersomes in order to enhance their cellular uptake. Fluorescein isothiocyanate labeled dextran containing polymersomes demonstrated that peptide linkers were cleaved in the lysosomal compartments of the cells, which led to membrane disruption [60]. The ability of enzyme-responsive polymersomes containing chemotherapeutic drugs or proteins to treat tumors is promising and remains to be tested [7].

13.2.4 Glucose-responsive polymersomes Glucose-responsive polymeric materials have received enormous attention in recent years because of their potential in the construction of closed-loop smart insulin delivery systems for the treatment of glucose-related human diseases including, type-1 and advanced type-2 diabetes [5, 7]. The incorporation of different glucose-sensing moieties into polymersomes has been based on three typical strategies, which include glucose oxidase (GOx), glucose-binding proteins, and boronic acids. The glucose-­ sensitive nanostructures can undergo structural transformations, namely shrinking, swelling, and dissociation, regulated by glucose concentration changes, leading to glucose-stimulated insulin release or glucose-mediated membrane permeability [5].

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Nanostructures containing boronic acid, especially phenylboronic acid (PBA), have been widely studied and used in the development of glucose-responsive materials for insulin delivery because of their rapid physicochemical changes in response to glucose. In general, at high dissociation constants (pKa ~8.5), PBA exists in equilibrium between the uncharged and charged forms, which are hydrophobic and hydrophilic forms, respectively. The PBA possesses the ability to form reversible complexes with 1,2-cis-diols of glucose, which result in the conversion of hydrophobic PBA into the hydrophilic form which might induce the release of entrapped molecules [61, 62]. In this regard, Kim’s group synthesized a glucose-responsive amphiphilic copolymer, composed of PEG and poly(styreneboroxole-alt-N-functionalized maleimide) (PEG-b-PBOx), using RAFT polymerization. The amphiphilic polymer could form polymersomes and loaded insulin effectively. Encapsulated insulin could be released from the polymersomes only in the presence of glucose under physiologically relevant pH conditions [63, 64]. Shi et al. prepared glucose-responsive polymersomes based on the complexation between a glucosamine (GA)-containing block copolymer PEG45-b-P(Asp-co-AspGA) and PBA-containing block copolymer PEG114-b-P(Aspco-AspPBA) with α-cyclodextrin/PEG45 inclusion complex as the sacrificial template. Vancomycin as a model drug was encapsulated in the polymersomes. From the release experiment, it was found that the drug was barely released in physiological buffer (pH 7.4). Interestingly, when glucose was added into the release medium, the vancomycin release was triggered [65]. On the other hand, 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 [66, 67], hypoxic [50, 51], or H2O2containing [47, 68] environment generated in the enzymatic reaction. Gu’s group constructed self-regulated insulin delivery nanovesicles by encapsulating GOx and insulin in pH-responsive polymersomes composed of PEG and ketal-­modified polyserine [66]. In those polymersomes, glucose can passively transport across their bilayer membrane for oxidation into gluconic acid by GOx, thereby causing a decrease in microenviroment pH. This local condition causes the hydrolysis of the pH-sensitive polymersome, which in turn triggers the release of insulin in a glucose-responsive manner. 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 this research group to construct a glucose-responsive insulin delivery device. Hypoxia and H2O2 dual-sensitive polymersomes were integrated into a painless microneedle array patch to provide fast insulin release and convenient administration (Fig. 13.11) [51]. Hubbell’s group loaded GOx into oxidation-responsive polymersome self-­ assembled by the synthetic amphiphilic block copolymer poly(ethylene glycol-b-­ propylene ­sulfide) (PEG-b-PPS). The enzymatic oxidation of glucose generates H2O2, which reacts with the PPS block, converting it into more hydrophilic sulfoxides and sulfones, which changed the hydrophilic-lipophilic balance of the macroamphiphile and thus inducing its solubilization and destroying the vesicles [69].

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Fig. 13.13  Schematic of the H2O2-responsive vesicles for glucose mediated insulin delivery. Self-assembly of block copolymer polyethylene glycol-phenylboronic ester-conjugated polyserine (mPEG-b-P(Ser-PBE)) into vesicles loaded with insulin and glucose oxidase (GOx). The vesicles are dissociated to release insulin in the presence of a hyperglycemic state. These polymersomes were further integrated into the hyaluronic acid-based microneedle-array patches for smart insulin delivery in a mouse model of type 1 diabetes. Reproduced with permission from X. Hu, J. Yu, C. Qian, Y. Lu, A.R. Kahkoska, Z. Xie, et al., H2O2-responsive vesicles integrated with transcutaneous patches for glucose-mediated insulin delivery, ACS Nano 11(1) (2017) 613–620, Copyright 2017 American Chemical Society.

Recently, Gu and coworkers reported a biodegradable and biocompatible H2O2 triggered glucose-responsive insulin delivery system by integrating H2O2-responsive polymersomes with transcutaneous microneedle-array patches to achieve fast response and painless administration. The polymersomes are self-assembled from block copolymer incorporated with PEG and phenylboronic ester (PBE)-conjugated polyserine (designated mPEG-b-P(SerPBE)) and loaded with glucose oxidase (GOx) and insulin. They showed usefulness as glucose sensing element (GOx) and act as reservoir of insulin to provide basal insulin release as well as promote insulin release in response to hyperglycemic states. In vivo studies indicated that a single patch can regulate glucose levels effectively with reduced risk of hypoglycemia (Fig. 13.13) [68].

13.2.5 Gas-responsive polymersomes Oxygen, carbon dioxide, nitric oxide, and hydrogen sulfide are typical biologically active gases that play important and fundamental roles in the human body because they are involved in intracellular biosignals. In recent times, the concept of using these gases as “green” stimuli to develop responsive polymersomes has received increasing attention as a topic of interest in biomimetic chemistry and polymersome research. Among them are CO2- or N2-responsive polymersomes, in which inactive CO2 or N2 gas is used

Stimuli-responsive polymersomes for drug delivery applications367

to regulate the self-assembly and disassembly of the nanostructures [70–73]. They have been study for different biomedical applications, such as bioimaging, diagnostics, and drug/gene delivery [30]. Yuan’s group reported a series of CO2-responsive polymersomes and their applications in shape transformation, smart surfaces, and controlled drug release. They successfully developed “breathing” 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 [71]. They synthesized the amphiphilic block copolymer poly(ethylene glycol)-b-poly((N-amidino)dodecylacrylamide) (PEG-b-PAD), which could self-assemble into polymersomes in aqueous solution. The size and volume of these polymersomes was tuned by alternating treatment with CO2 and Ar through the protonation and deprotonation effect [72]. 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 self-expansion of the nanostructures. By regulation of the CO2 simulation time, the growth of polymersomes and membrane permeability can be tuned, which is useful for controlling payload release and selective separation of different guest molecules. These polymersomes with unique gas-responsiveness were shown to have reversible distinctive expansion and contraction; therefore, they can be regarded as functional “breathing” nanocontainers for periodically accelerating drug release (Fig. 13.14) [71, 72]. Also, this system can also be used as a nanoreactor for enzymatic catalytic reactions [5]. Zhao’s group used the same mechanism to observe CO2-driven self-assembly and shape transformation of PEO-b-PAD with a broad range of shapes, from microscopic tubules to submicroscopic vesicles and nanomicelles by modulation of the CO2 level, which adjusted the copolymer hydrophilic-hydrophobic ratio [74]. This group also reported CO2-sensitive glycopolypeptide polymersomes assembled from two end-­decorated biopolymers, dextran-ß-cyclodextrin (Dex-CD), and poly(l-valine)-­ benzimidazole (PVal-b-Bzl), which show a reversible assembly and disassembly process that can also be tuned by CO2, biomimicking virus capsids [75]. Another type of CO2-responsive materials is amine- or carboxylic acid-­ containing polymers. Zhao’s group synthesized poly(ethylene oxide)-b-polystyreneb-­ poly((2diethylamino)ethyl methacrylate) (PEO-b-PS-b-PDEAEMA) triblock ­copolymers [76] and studied their gas-triggered shape transformation. The tertiary amine groups in PDEAEMA can react with CO2 in water, showing an extended hydrophilic chain conformation, and this process is reversible upon exposure to N2 to remove CO2 [77]. Three initial nanostructures, including spherical micelles, wormlike micelles, and vesicles, were obtained by varying the length of the PS block, while the PDEAEMA blocks always constituted the inner CO2-responsive part of the core. Different CO2-controlled deformations, such as volume expansion of spherical nanostructures, stretching of curly nanofibers, and compartmentalization of vesicles, were achieved through CO2-induced protonation of the pendant tertiary amine in the PDEAEMA block. The authors reported two other CO2-responsive vesicles based on the block copolymers PDMA-b-PDEAEMA (poly(N,N-­dimethylacrylamide), PDMA) and PEO-b-P(DEAEMA-co-CMA) (coumarin, CMA) using the same m ­ echanism.

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Fig. 13.14  (A) (a) Gas-switchable chemical structural change of the poly(ethylene glycol)-bpoly((N-amidino)dodecylacrylamide, PEG-b-PAD) block copolymer. (b) Self-assembly of the copolymer into polymersomes and reversible gas-controlled breathing behavior in aqueous media. (B) Schematic representation 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; and (c) after 30 min of CO2 exposure. Reproduced with permission from X. Hu, Y. Zhang, Z. Xie, X. Jing, A. Bellotti, Z. Gu, Stimuli-responsive polymersomes for biomedical applications, Biomacromolecules 18(3) (2017) 649–673, Copyright 2017 American Chemical Society.

For PDMA-b-PDEAEMA polymersomes, morphological changes (from expansion to bubbling) as a result of protonation of the DEAEMA units in the vesicle membrane were observed. PEO-b-P(DEAEMA-co-CMA) polymersomes containing crosslinked 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 [72]. Yuan and coworkers further combined CO2-sensitive PDEAEMA with temperature-­responsive PNIPAM to obtain a CO2- and temperature-switchable “schizophrenic” block copolymer. With a simple synthetic strategy, the authors have successfully exploited the use of CO2 and temperature to trigger schizophrenic micelle to vesicle morphological transition of polymer assemblies. They anticipated that “green” stimuli methods to realize unique self-assembly behavior in dilute aqueous solution may offer new possibilities in

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“on-demand” release or absorption of chemicals, ­including drugs. As both CO2 and temperature play a key role for living organisms, the system developed also provides inspiration for biomimicry by using synthetic polymers [78].

13.2.6 Temperature-responsive polymersomes Temperature is a popular stimulus that can be used to trigger the specific responsiveness of polymersomes. Temperature-responsive polymers exhibit a phase transition at a certain temperature, which results in changes in conformation, solubility, and hydrophilic-hydrophobic balance. Polymers which become soluble upon heating have a so-called upper critical solution temperature (UCST), and those ones which become insoluble upon heating are characterized by a lower critical solution temperature (LCST). This property has been used in several ways in biomedical applications. In particular, for drug delivery, the release of a drug could be in response to an endogenous temperature increase that makes the thermosensitive polymer collapse, or to an externally applied temperature increase [79]. This is particularly interesting in cancer therapy due to the local temperature is slightly higher in solid tumors than normal body; then, by adjusting the LCST of the thermosensitive polymer to be between body temperature and the higher temperature of the tumor, it is possible for the drug delivery systems to accumulate into the tumor [7, 79]. Temperature-responsive nanocarriers have started to be explored recently. Unfortunately, the most thoroughly studied copolymer systems are relatively temperature insensitive. Although, a wide range of temperature-responsive block copolymers have been synthesized, PNIPAM is the most used block to synthesize temperature-­ sensitive copolymers [5, 30, 79]. In particular, PNIPAM can transit between hydrophilic to hydrophobic when the temperature is switched around its LCST of 32°C, which is just below the physiological body temperature. When PNIPAM is applied as the hydrophobic part of a polymer system, it can self-assemble to form stable polymersomes at the normal body temperature of 37°C (higher than the LCST), while disassemble and rapidly release encapsulated molecules at temperatures below 32°C [30]. As it was mentioned, thermally triggered assembly/disassembly of these polymers can be exploited for drug delivery or injectable gelation. Qin et al. used the diblock copolymer PEO-b-PNIPAM, which is amphiphilic in water above body temperature and can self-assemble into polymersomes, encapsulating both hydrophilic drugs in the aqueous lumen and hydrophobic molecules in the membrane. With a decrease in temperature (below 32°C), the PNIPAM block becomes hydrophilic, and the vesicles disassemble allowing temperature-controlled quick release of both types of loaded molecules [80]. The PNIPAM block has also been conjugated to hydrophobic polymer blocks, and the resultant copolymers formed assemblies at room temperature that were further aggregate into complex morphologies at high temperatures [5]. Moughton and O’Reilly described a diblock copolymer (PtBuA-b-PNIPAM) in which the PNIPAM block had a permanently hydrophilic charged quaternary amine “head-group,” They reported a thermally induced micelle to vesicle morphology transition induced morphology transition from micelles to vesicles due to charged chain end diblock copolymer. They proposed that developed vesicles may show limited

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membrane permeability toward hydrophilic, positively charged species which could facilitate their use in temperature-responsive hydrophilic scavenger/encapsulation applications. Also, they postulated that the exterior and interior charged “head-group” functionality could be exploited for the binding of biologically relevant species such as proteins, DNA, or RNA [81]. Huang et al. conjugated PNIPAM to proteins to obtain temperature-responsive microscale compartments called “proteinosomes,” which are delineated by a semipermeable, stimulus-responsive, enzymatically active, elastic membrane consisting of a closely packed monolayer of conjugated protein-polymer building blocks. Proteinosomes exhibited protocellular properties such as guest molecule encapsulation, selective permeability, protein synthesis via gene expression, and membrane-gated internalized enzyme catalysis. The researchers demonstrated the thermally induced gating of membrane permeability to external substrates, which effectively produces an on/off switch for enzymatic reactions inside the microscale compartments (Fig. 13.15) [82]. Dual or multi-responsive polymersomes, mainly those combining sensitivity to temperature and reduction, temperature and pH, or temperature and light, have been often investigated. For instance, McCormick and coworkers developed temperature-­ responsive nanostructures based on poly(N-(3-­aminopropyl)methacrylamide hydrochloride)-b-­PNIPAM (PAMPA-b-PNIPAM) and poly(2(dimethylamino) ethylmethacrylate)-­b-PNIPAM (PDMAEMA-b-PNIPAM) dispersed in water [83, 84]. The hydrophilic PAMPA block was ionically cross-linked through the addition of an oppositely charged polyelectrolyte. The PDMAEMA-b-PNIPAM-based polymersomes were stabilized by the reduction of NaAuCl4 to form hybrid aggregates with Au nanoparticles incorporated in the PDMAEMA domain. After cross-linking, the polymersomes were “locked” in place and could dissociate only upon swelling when the temperature was lowered. These polymersomes showed promising properties as smart carriers for triggered intracellular gene and drug delivery. In addition, Peng’s group prepared temperature-responsive vesicles based on poly(2-cinnamoylethyl methacrylate)-­b-poly(N-isopropylacrylamide) (PCEMA-b-PNIPAM) [85, 86]. The outer and inner surfaces of the nanostructures undergo a reversible coil-globule transition upon changing the temperature of the system [85]. The PCEMA-b-PNIPAM were subsequently photo-cross-linked the PCEMA shells. In those vesicular aggregates, PCEMA chains formed a hydrophobic shell and help to “lock in” the structure of the aggregates by photo-cross-linking, while PNIPAM chains stretched into the aqueous phase from both the outer and inner surface of the hydrophobic shell in order to stabilize the vesicles. Also, PNIPAM chains exhibited a reversible thermo-responsive phase transition at LCST in aqueous solution, which may provide on/off switches for the polymersomes [86]. Zhong’s group prepared PEG-b-PAA-b-PNIPAM polymersomes by heating polymer solutions to 40°C and cross-linking the PAA segments with reduction-sensitive cystamine 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. Various proteins including BSA, lysozyme, cytochrome C, and ovalbumin could be conveniently loaded into the polymersomes with markedly high protein loading efficiencies. The in  vitro release studies using

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Fig. 13.15  Procedure for the preparation of proteinosomes based on surface primary amine groups of cationized proteins such as bovine serum albumin (BSA-NH2) and poly(Nisopropylacrylamide) (PNIPAM). (A) Coupling of mercaptothiazoline-activated PNIPAM polymer chains with primary amine groups of cationized (BSA-NH2) to produce proteinpolymer nanoconjugates (BSA-NH2/PNIPAM). (B) Use of protein-polymer building blocks for the spontaneous assembly of proteinosome micro-compartments in oil, and their transfer into a bulk water phase. (C) Schematic illustration showing the procedure for cell-free gene expression of enhanced green fluorescent protein (eGFP) and images of them obtained by optical (left) and fluorescence (right) microscopies (scale bar, 100 μm). Reproduced with permission from X. Huang, M. Li, D.C. Green, D.S. Williams, A.J. Patil, S. Mann, Interfacial assembly of protein–polymer nano-conjugates into stimulus-responsive biomimetic protocells, Nat. Commun. 4 (2013) 2239, Copyright 2013 Springer Nature.

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cystamine-cross-­linked polymersomes showed that release of payload was minimal (ca. 20%) in 11 h in PBS at 37°C, while fast protein release of over 70% was observed under an intracellular mimicking reductive environment [42, 87]. Other temperature-sensitive polymers, such as poly(propylene oxide)-b-PLys [88], poly(trimethylene carbonate)-b-PGA [89], poly(trans-N-(2-ethoxy-1,3dioxan-5-yl) acrylamide) [90], modified poly(aspartamide) [91, 92], and poly(N-vinylcaprolactam) (PVCL) [93] have also been reported. As an example, Kharlampieva’s group reported polymersomes composed of a novel type of temperature-sensitive triblock copolymer PVCL-b-polydimethylsiloxane-b-PVCL (PVCL-b-PDMS-b-PVCL), which showed temperature-controlled permeability within the physiologically relevant temperature range of 37°C–42°C for sustained delivery of anticancer drugs. The copolymers assembled into stable vesicles at room temperature. Remarkably, the permeability of polymersomes loaded with the anticancer drug doxorubicin could be precisely controlled by PVCL length in the temperature range of 37°C–42°C. At elevated temperatures, above the LCST of PVCL, transient pores created in the PVCL-b-PDMS-b-PVCL membrane allowed drug to easily pass through the hydrophobic PDMS layer without damaging the vesicles (Fig. 13.16). Moreover, the polymersomes were biocompatible, biodegradable, monodisperse, and stable at room temperature, with tunable size and thermal responsiveness provided by amphiphilic triblock copolymers [93]. On the other hand, pH-responsive tertiary amines containing polymers usually have temperature-dependent pKa and consequently have been integrated into polymersomes to use this temperature sensitivity. In this sense, Agut et al. prepared the p­ olypeptide-based

Fig. 13.16  Poly(N-vinylcaprolactam)-b-polydimethylsiloxane-b-poly(N-vinylcaprolactam) (PVCL-b-PDMS-b-PVCL) vesicles formed at room temperature decrease in size and become permeable to doxorubicin at temperatures from 37°C to 40°C. Reproduced with permission from F. Liu, V. Kozlovskaya, S. Medipelli, B. Xue, F. Ahmad, M. Saeed, et al., Temperature-sensitive polymersomes for controlled delivery of anticancer drugs, Chem. Mater. 27(23) (2015) 7945–7956, Copyright 2015 American Chemical Society.

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Thermo-responsive micelles

DPPGA

High temperature

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DPPGA DPPGA

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186

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77

Insoluble complexes

77 37

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2

3

Electrostatic vesicles

4

5

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6

7

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Fig. 13.17  Schematic representation of the different morphologies obtained from polypeptidebased multiresponsive block copolymers. Reproduced with permission from W. Agut, A. Brûlet, C. Schatz, D. Taton, S. Lecommandoux, pH and temperature responsive polymeric micelles and polymersomes by self-assembly of poly [2-(dimethylamino) ethyl methacrylate]-b-poly (glutamic acid) double hydrophilic block copolymers, Langmuir 26(13) (2010) 10546–10554, Copyright 2010 American Chemical Society.

double hydrophilic block copolymer PDMAEMA-b-PGA and investigated its pH- and temperature-responsiveness involved in their self-assembly behavior. They demonstrated that the process of self-assembly into polymersomes and micelles could be tuned as a function of pH and/or temperature and proposed these possibilities of variation in size and shape of morphologies as useful strategies in the development of multiresponsive nanocarriers for biomedical applications (Fig. 13.17) [94]. Pearson et al. investigated the effect of temperature on a pH-responsive amphiphilic diblock copolymer, namely poly(2-(methacryloyloxy)ethyl phosphorylcholine)-­poly(2(diisopropylamino)ethyl methacrylate) (PMPC-b-PDPA). They observed that the pH-modulated amphiphilic character of PMPC-b-PDPA drives its self-assembly in aqueous solution [95]. In addition, poly(2-vinylpyridine) [96] and PDMA-b-PS-b-poly(N-(4vinylbenzyl)-N,N-diethylamine) [97] are other tertiaryamine-based temperature-­sensitive polymers and their morphology transitions have also been investigated.

13.2.7 Photo-responsive polymersomes Light is a remote stimulus that does not require the addition of external triggers or sensitive moieties. Photo-responsive self-assembled nanostructures have received great attention because the responsiveness of the assemblies can be rapidly and conveniently induced at a specific time and location upon exposure to wavelengths which can be

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visible light, ultraviolet, and near-infrared (NIR). These systems are often based on the principle that photo-sensitive moieties are incorporated in polymers that function as light-cleavable linkers, undergo light-induced degradation or light-responsive conformational changes. Light, an external source used as a trigger, has been used for the design of intelligent delivery systems because the release of entrapped therapeutic agents, can be rapidly induced at specific time points and locations by exposure to light in an on/off switching manner. The release profiles of bioactive molecules from such polymersomes can be regulated by adjusting the light wavelength, intensity, and exposure time. The absorbance of light in the window of 650–900 nm, in the NIR, is particularly interesting for biomedical applications due to minimum absorbance by tissue and skin. For these reasons, light-responsive polymersomes have potential for on-demand drug delivery and noninvasive clinical therapy. To prepare photo-­ responsive polymersomes, photo-sensitive moieties have to be incorporated into block copolymer. Once obtained, the polymersomes must be disrupted and dissociated based on photoinduced polymer degradation and/or cleavage of block junctions, or photoinduced structural and/or property changes, including the hydrophobic-hydrophilic balance and reversible photo-cross-linking. The most widely reported photo-responsive moieties include azobenzene (AZO), spiropyran (SP), 2-diazo-1,2-naphthoquinone (DNQ), o-nitrobenzyl (ONB), and coumarin derivatives are the most widely reported photo-responsive moieties incorporated into the amphiphilic polymer systems to make them susceptible to light [5, 7, 30]. The ONB derivatives have been investigated as photo-cleavable photochromic molecules, which are positioned in the main chain, side chain, or block junction of block copolymers and breaks the hydrophobic-hydrophilic balance of the system, inducing the degradation of the backbone into oligomers under irradiation. Cabane et  al. synthesized an amphiphilic block copolymer containing a photodegradable linker as a junction point between hydrophilic PAA and hydrophobic ONB-substituted poly(γ-methyl-ε-caprolactone) (PMCL-ONB) blocks. The vesicles disintegrated upon UV irradiation, yielding small micellar-like structures and simultaneously releasing the preloaded low-molecular-weight dye and proteins. The copolymers can self-assemble into different structures, including micelles and vesicles which are photo-responsive. When polymer dispersions were exposed to UV irradiation, the vesicles were disintegrated yielding small micellar-like structures and simultaneously releasing the preloaded low-molecular-weight dye and proteins. Moreover, the payload was released in a controlled manner by varying the UV intensity (Fig.  13.18) [98, 99]. Burdick’s group reported a novel route for the synthesis of PCL-b-PEG diblock copolymers that allows for the insertion of functional groups at the block junctions and the assembly of functional membranes. They incorporated an amino acid functional group at the junction to develop the photo-cleavable polymersomes. 2-­nitrophenylalanine was used to create UV-responsive membranes in which the vesicles were destabilized and released encapsulated contents upon irradiation [100]. Liu’s group reported self-immolative polymersomes self-assembled from amphiphilic block copolymers consisting of a triggered degradable poly(benzyl carbamate) (PBC) block and a hydrophilic PDMA block. The hydrophobic blocks exhibited stimuli-triggered

Stimuli-responsive polymersomes for drug delivery applications375

(A)

Photocleavable linker Hydrophilic + Hydrophobic

NO2

O Br

O

16 O

OH

O O

Br

H

16 O

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Drug

H

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Poly(acrylic acid)16-O-Nitrobenzyl-poly(methyl caprolactone)76 PAA16-ONB-PMCL76

(B)

NO

O

O

O

+

HO

O O

H 76

Degradation products

PAA ONB PMCL

Fig. 13.18  (A) Schematic illustration of the amphiphilic photocleavable block copolymer, chemical structure of the poly(methyl caprolactone)-ONB-poly(acrylic acid) (PMCL-ONBPAA) diblock copolymer, and its degradation products upon UV irradiation. Upon cleavage, PAA chains bearing the photo-degraded linker and PMCL chains bearing -COOH end groups are formed. (B) Scheme depicting polymersomes, and the conformation of the assembled polymer chains forming their membranes. Upon UV exposure (second step), the corona PAA chains are cleaved, that is, separated from the PMCL, forming the core of the membrane. Consequently, the vesicle membrane is destroyed (third step) and the payload released. Reproduced with permission from E. Cabane, V. Malinova, S. Menon, C.G. Palivan, W. Meier, Photoresponsive polymersomes as smart, triggerable nanocarriers, Soft Matter 7(19) (2011) 9167–9176, Copyright 2011 The Royal Society of Chemistry.

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head-to-tail cascade depolymerization features. Different triggers, including visible light, UV light, or a reductive milieu could be utilized to activate polymersomes disintegration into water-soluble small molecules and hydrophilic blocks [101]. In addition to the most reported vesicle dissociations or vesicle-to-unimer transitions upon irradiation, Liu’s group also prepared UV-regulated “traceless” cross-linked polymersomes. 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 self-immolative decaging reactions release primary amine moieties and extensive amidation reactions. This leads to vesicle cross-linking and the process is associated with bilayer hydrophobicity-to-hydrophilicity transition and membrane permeabilization [102]. On the other hand, the AZO moiety can reversibly transform from the trans isomer to the cis isomer upon visible and UV light irradiation. This behavior has been investigated for triggering the disruption and disassembly of AZO-containing polymersomes in a reversible manner [103, 104]. Su et  al. synthesized a block copolymer composed of PAA as the hydrophilic block and poly[6-(4-(4-methylphenylazo) phenoxy) hexyl acrylate], an AZO-containing polyacrylate, as the hydrophobic block (PAA-b-PAzoM), which self-assembled into giant spherical microvesicles in a mixture of water and tetrahydrofuran (THF). Upon irradiation with 365 nm light, the AZO side groups underwent trans-to-cis isomerization that induced a deformation of the vesicles from a spherical shape to an earlike shape [105]. Also, this research group developed photo-responsive polymersomes from block copolymers PNIPAM-b-PAzoM which also self-assembled into giant microvesicles in a mixture of water and THF. Fusion of these self-assemblies was observed upon irradiation with 365 nm light and the real-time process was observed under an optical microscope [106]. Yu’s group reported photo-responsive polymersomes based on a block copolymer consisting of hydrophilic PEG and hydrophobic azopyridine-containing polymethacrylate (PAP). The nanostructures underwent photoinduced circular process including fusion, damage and defect formation, disruption, disintegration, and rearrangement in water and THF mixtures upon exposure to UV light. The process of photo-responsive cycle can be inhibited at any moment by visible light [103]. Systems with other types of architectures, such as host-guest interactions and dendritic block copolymers have also been explored. Jin et  al. prepared the AZOcontaining block copolymer PEO-b-poly(6-(4-phenylazophenoxy)hexyl methacrylateco-­2(dimethylamino)ethyl methacrylate) (PEO-b-P(AzoMA-co-DMAEMA)), which assembles into vesicles in water. Alternating irradiation of the solution with UV and visible light induced the reversible supramolecular self-assembly and disassembly of vesicles because of the photoinduced trans-to-cis isomerization of AZO units. Photostimuli control the inclusion and exclusion reactions of ß-cyclodextrin and AZO, therefore enabling reversible photo-responsive self-assembly and disassembly based on the wavelength of the irradiating light (365 or 450 nm) [107]. Xia et al. reported photo-responsive self-assemblies in water based on the photo-responsive recognition motif between a water-soluble pillar[6]arene host and an AZO-containing amphiphilic guest. The guest itself self-assembled into solid nanoparticles before complexation with the host. A reversible transition between vesicles and solid nanoparticles was

Stimuli-responsive polymersomes for drug delivery applications377

achieved with the trans-to-cis photo-isomerization of the AZO groups upon application of UV and visible light [108]. Blasco et  al. 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-­isobutyloxy-AZO units and hydrocarbon chains (C18) randomly connected to the periphery of the dendron. The influence of AZO/C18 ratio in the photo-response of the self-assemblies was assessed as well as the encapsulation of both hydrophilic (Rhodamine B) and hydrophobic (Nile Red) fluorescent probes and the use of light as an external stimulus to trigger the release of the probes. The self-assembly and light-responsiveness of the vesicles were explored. Both hydrophilic (Rhodamine B) and hydrophobic (Nile red) compounds were encapsulated into the vesicles, and the photo-triggered drug release and controlled polymer degradation were studied [109–111]. In addition, the use a SP, a well-known photochromic molecule, has been also evaluated. SP can undergo reversible isomerization between the hydrophobic ring-closed SP form and the hydrophilic ring-opened merocyanine (MC) form under UV and visible light irradiation in a wavelength-selective manner. The photo-tunable SP-to-MC isomerization process has been exploited to reversibly regulate the permeability of polymersomes. Wang et al. reported photochromic polymersomes exhibiting photo-switchable and reversible bilayer permeability from newly designed PEO-b-PSPA diblock copolymers, where SPA is SP-based monomer containing a unique carbamate linkage. Upon self-assembling into polymersomes, SP moieties within vesicle bilayers undergo reversible photo-triggered isomerization between hydrophobic SP (λ2 > 450 nm irradiation) and zwitterionic MC (λ1 < 420 nm irradiation) states. Also, reversible photo-triggered SP-to-MC polymersome transition is accompanied by membrane polarity and permeability switching from being nonimpermeable to selectively permeable toward noncharged, charged, and zwitterionic small molecule species below critical molar masses, where switchable drug release triggered by alternating exposure to UV and visible light could be observed [112]. Zhou et al. prepared block copolymer free hyperbranched supramolecular amphiphiles through the noncovalent coupling of functionalized adamantane or AZO with hyperbranched polyglycerol grafted ß-cyclodextrin [113, 114]. The functionalized linear-hyperbranched supramolecular amphiphile self-assembled into vesicles with a narrow size distribution, and the vesicles can be disassembled readily by the introduction of competitive hosts. The Janus hyperbranched polyglycerol prepared by AZO/ß-cyclodextrin complexation between ß-cyclodextrin-g-hyperbranched polyglycerol could form vesicles in an aqueous solution [114]. As it was mentioned, the AZO group could undergo light-triggered reversible isomerization between the trans and the cis form under UV light irradiation; as a result, the Janus hyperbranched vesicles could be disassembled under irradiation of UV light (365 nm). This kind of system could be useful for pulsatile drug delivery applications. Most of the abovementioned photo-responsive self-assemblies are based on irradiation with visible and UV light. Although various UV-responsive polymersomes have been shown to possess excellent properties in vitro, this radiation is only applicable for the treatment of certain regions of the human body that can be directly irradiated, such as the eye or the skin, which limits the biomedical applications of these

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responsive polymersomes. For certain clinical applications, NIR-light-sensitive polymersomes are ideal and promising candidates because of advantages including deep tissue penetration, low scattering loss, and minimal harm to tissues [5, 7]. Some NIRphoto-responsive polymersomes have been developed by incorporating chromophores that can respond to long wavelengths or exploit two-photon technology. One of these strategies uses the encapsulation of NaYF4:TmYb (a rare-earth-doped) in upconversion nanoparticles for NIR-triggered drug release and photo-switching. When irradiated with NIR light, the upconversion nanoparticles can absorb the light (980 nm) 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 by coupling to an organic photosensitizer. That strategy of using upconversion nanoparticles as an internal UV or visible light source upon NIR light excitation represents a general and efficient method to circumvent the need for UV or visible light excitation that is a common drawback for light-responsive polymeric systems developed for potential biomedical applications [115, 116]. A related strategy involves NIR-absorbing plasmonic materials, such as metal nanoparticles or organic chromophores, to convert photon energy to heat which trigger the release of bioactive molecules from NIR-photo-responsive polymersomes. This process is called photothermal effect and it has been investigated for biomedical applications [117–119]. In this sense, Nie’s group prepared a multifunctional theranostic platform based on photosensitizer (Ce6)-loaded plasmonic vesicular assemblies of gold nanoparticles for effective cancer imaging and treatment. The Ce6-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 photo-responsive polymersomes showed strong NIR absorption and the capability of encapsulating the photosensitizer Ce6 in gold vesicle enable trimodality NIR fluorescence, thermal and photoacoustic imaging-guided synergistic photothermal, and photodynamic therapy of tumors in vivo (Fig. 13.19) [118, 120].

13.2.8 Magnetic field-responsive polymersomes Magnetic field-responsive systems have promising biomedical applications in therapeutics, imaging, and diagnostics because of their noninvasive nature, high penetration, absence of energy dissipation, and ease of control. 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). Magnetic field-responsive systems are susceptible to magnetic guidance, induced temperature increase, or a combination of both [5, 7, 30]. Lecommandoux and coworkers have studied polymersomes that self-assemble from the block copolymer PTMC-b-PGA and encapsulate both ultrasmall superparamagnetic iron oxide (γFe2O3) nanoparticles (USPIO NPs) and doxorubicin within the membrane. The researchers studied the magnetic field-triggered drug release and the deformation of the vesicle membranes caused by local hyperthermia under an applied magnetic field [15, 121]. This research group also studied other self-assemblies based on PBD-b-PGA diblock copolymers, which were used to prepare magnetic micelles and vesicles [122].

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Fig. 13.19  Photosensitizer (Ce6)-loaded plasmonic gold vesicles (GVs) for trimodality fluorescence/thermal/photoacoustic imaging guided synergistic photothermal/photodynamic cancer therapy. Reproduced with permission from J. Lin, S. Wang, P. Huang, Z. Wang, S. Chen, G. Niu, et al., Photosensitizer-loaded gold vesicles with strong plasmonic coupling effect for imaging-guided photothermal/photodynamic therapy, ACS Nano. 7(6) (2013) 5320–5329, Copyright 2013 American Chemical Society.

Förster’s group prepared vesicles loaded with Fe3O4 nanoparticles incorporated into the bilayer membrane of poly(2-vinylpyridine-b-ethylene oxide) (P2VP-b-PEO) polymersomes. The hydrophobic nanoparticles were not located in the center of the bilayer but rather at the periphery decorating the hydrophobic/hydrophilic interface because of the low entropy of mixing between particles and polymers. The loaded Fe3O4 nanoparticles resulted in a tendency to bridge to adjacent bilayers leading to the formation of oligo- and multilamellar vesicles [123]. Gong’s [124] and Du’s groups [125] prepared multifunctional SPIO/doxorubicin-loaded polymer vesicles to evaluate their potential use in targeted cancer therapy and MRI. Hickey et al. reported the self-assembly of magnetic nanoparticles based on the amphiphilic block copolymer PAA-b-PS and their controlled morphology with magnetic nanoparticles distributed in different regions of the assembly [126]. Mart et al. prepared Fe3O4 nanoparticles-­ vesicle assemblies embedded within a hydrogel extravesicular matrix and demonstrated remote magnetic-triggered payload release [127].

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Most of the reported magnetic field-responsive systems were constructed by encapsulation of magnetic nanoparticles into polymersomes. Magnetic field-responsive systems can also be developed using diamagnetic structures assembled from amphiphilic block copolymers for magnetic manipulation. For instance, van Hest’s group used diamagnetic structures assembled from amphiphilic block copolymers for magnetic manipulation. They prepared bowl-shaped polymer stomatocytes by highly regulating the shape of PEG-b-PS amphiphilic block copolymers. They found that the size of stomatocytes opening increased along with increasing the intensity of the magnetic field and this process was reversible when the magnetic field was removed. The mechanism of magnetic field-modulated opening of the stomatocytes was caused by the highly anisotropic magnetic susceptibility of the building blocks, PEG-b-PS, which resulted in their collective perpendicular alignment in the magnetic field. This led to stretching of the membrane, resulting in a deformation of the structure and increase in the opening. They then studied the capture and release of various payloads using this controlled opening/closing mechanism under magnetic fields (Fig. 13.20) [128–131].

Fig. 13.20  Schematic representation of the strategy for capture and release of cargo with stomatocyte magneto-valves. Reproduced with permission from P. Van Rhee, R. Rikken, L. Abdelmohsen, J. Maan, R. Nolte, J. Van Hest, et al., Polymersome magneto-valves for reversible capture and release of nanoparticles, Nat. Commun. 5 (2014) 5010, Copyright 2014 Springer Nature.

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13.2.9 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. Ultrasound, an external stimulus, is used as an adjuvant of chemotherapy in tumor treatment. In chemotherapy, ultrasound can be used as a sensitizer to enhance chemotherapy and overcome drug resistance. Compared with light, which does not have time and target selectivity, ultrasound is effective for deep tissue penetration by tuning the frequency, duty cycles, and time of exposure [5, 7]. Zhou et al. synthesized air-encapsulated PEG-b-PLA polymersomes, and under a medical ultrasound, polymersome bubbles were visualized as bright spots using a medical ultrasound scanner. These air-containing polymersomes have potential applications in targeted ultrasound imaging and triggered drug release [132]. Chen and Du reported novel polymersomes that are responsive to both ultrasound and pH (Fig. 13.21). The polymersomes were prepared from a triblock copolymer based on PEG-b-­poly[2(diethylamino)ethyl methacrylate-stat-tetrahydrofuranyloxy)ethylmethacrylate] (PEGb-p(DEA-stat-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. In response to 180 W ultrasound radiation, the vesicles shrank to accelerate doxorubicin release. Upon ultrasound exposure, the PTMA chains hydrolytically reduced and the hydrophobic balance was altered, which resulted in the shrinking of the polymersomes [133].

O O

O 43

Br 33 O

O

PEO DOX×HCl PTMA PDEA

47 O

N

O

DOX×HCl

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Self-assembly in THF/water at pH 7.4

O

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pH < 4.8 faster release

Full protonation of PDEA results in disassembly of vesicle

4.8 < pH < 7.3 faster release

Recrystallization of PTMA surpassing protonation of PDEA results in smaller vesicle

Ultrasound faster release

Drug-loaded pH and ultrasound dually responsive vesicle

Ultrasound disruption and re-self-assembly result in smaller vesicle

Fig. 13.21  Preparation of ultrasound and pH dual-responsive polymersomes from PEG-bP(DEA-stat-TMA) copolymers and their drug release triggered by lowering the pH or by ultrasound radiation. Reproduced with permission from W. Chen, J. Du, Ultrasound and pH dually responsive polymer vesicles for anticancer drug delivery, Sci. Rep. 3 (2013) 2162, Copyright 2014 Springer Nature.

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13.2.10 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 interesting applications in biological systems [5]. Recently, the preparation of block copolymer-free reversible nanostructures has received enormous attention because of the easy fabrication and tunable structural properties of these materials. Block copolymer-free polymersomes can be obtained via host-guest interactions between the end group of hydrophilic and hydrophobic homopolymers [134]. Cyclodextrins, composed of cyclic oligosaccharides with high biocompatibility, are known for their ability to form inclusion or host-guest complexes with various hydrophilic and lipophilic molecules via hydrogen bonding or hydrophobic interactions [135]. Yin’s group prepared electric field-responsive vesicles based on chain end-decorated poly(styrene)-β-cyclodextrins (PS-β-CD) and PEG-ferrocene (PEG-Fc) homopolymers (Fig. 13.22). These materials form reversible diblock copolymers (PS-ß-CD/PEG-Fc) via the host-guest interaction between CD and Fc. Under aqueous conditions, the copolymers self-assembled into vesicular structures. The application of +1.5 V electrochemical stimuli initiated the disassembly of the vesicles. The voltage-responsiveness of the polymersomes was further verified by the polymersomes with Rhodamine B, where its release was precisely tuned by varying the applied potential. The unique features of voltage-­responsive polymersomes offer a new approach for stimuli-responsive drug delivery; such systems are well suited for applications that require structural changes without the addition of chemical reagents [136].

PS-b-CD 9.7 nm

+

PEO-Fc 4.6 nm

Orthogonal Assembly

PS-b-CD/PEO-Fc 13.1 nm

Self-assembly in water PEO-Fc +

+1.5V 19 nm

–1.5V

Loaded molecules

Fig. 13.22  Structure of poly (styrene)-ß-cyclodextrins (PS-ß-CD) and poly(ethylene oxide)ferrocene (PEO-Fc) and schematic of the voltage-responsive controlled assembly and disassembly of PS-ß-CD/PEO-Fc supramolecular vesicles. Reproduced with permission from Q. Yan, J. Yuan, Z. Cai, Y. Xin, Y. Kang, Y. Yin, Voltageresponsive vesicles based on orthogonal assembly of two homopolymers, J. Am. Chem. Soc. 132(27) (2010) 9268–9270, Copyright 2010 American Chemical Society.

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Although PS-ß-CD/PEG-Fc-based polymersomes showed promising voltage-­ responsive properties, their disassembly in aqueous solutions caused the formation of hydrophobic aggregates due to the hydrophobic PS homopolymer. To overcome this limitation, Park’s group et  al. developed electric field-responsive polymersomes using the redox responsiveness of an amphiphilic rod-coil molecule, tetraaniline poly(ethylene glycol) (TAPEG). In aqueous solutions, the amphiphile TAPEG in its reduced leucoemeraldine base (LEB) form self-assembles into unilamellar vesicles and effectively encapsulated a fluorescein derivative. Upon application of an oxidizing voltage the vesicle membrane splits into smaller micellar structures (puck-like micelles), which then reassemble to form vesicles upon exposure to a reducing voltage. These electrically switchable vesicles were also exploited for molecular delivery [135].

13.3 Remarks and future perspectives The crucial aim of research in nanocarriers for biomedical applications is to develop effective and safe treatments for clinic use. The development of polymersomes with increased efficacy and reduced adverse effects is particularly important for improving therapies. In this chapter, it were highlighted several representative examples of stimuli-responsive polymersomes, which are the most promising nanostructures for biomedical applications. Polymersomes are self-assembled amphiphilic polymers in which an aqueous compartment is enclosed by a thick bilayer membrane. Unlike conventional polymersomes, stimuli-responsive polymersomes rapidly release drugs at the target site in response to a specific stimulus. The introduction of stimuli-­triggered responsiveness allows polymersomes to recognize variations external physical or internal biological environment and conduct “on-demand” release in dose-, spatial-, and temporal-controlled fashions. Stimulus such as pH, redox, enzymes, temperature, light, magnetic fields, electric fields, and ultrasound, have been used to disrupt the hydrophobic-hydrophilic balance of polymersomes to destabilize their assemblies. These types of stimulus can lead conformational change in the structure of the polymersomes and the payload would immediately be released. Numerous reports demonstrate the broad research in the development of stimuli-­ responsive polymersomes. Advances in polymer synthesis allow design and development polymersomes with tailorable physicochemical, pharmacological, and biological properties. Also, cross-linking or targeting moieties allow for improved in vitro and in vivo stability, prolonged circulation time, and enhanced accumulation at the target sites. Tunable membrane permeability and incorporation of specific channel-forming proteins have been studied for cell mimics. Nevertheless, there is a lot of work to do forward. Many scientific and engineering issues must be addressed for the successful translation of basic research to clinical applications. More and advanced in vivo studies need to be done to better understand of the interactions between polymersomes and living organisms. The biocompatibility and safety of polymersomes should be studied. Most of the aforementioned reports regarding stimuli-responsive polymersomes showed that the nanostructures were evaluated in  vitro and/or in small animals. Even though polymersomes showed minimal toxicity, long-term toxicity, and immunogenicity studies

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should be performed, especially when the biomedical applications require systemic administration. Furthermore, most stimuli-responsive studies have been demonstrated under relatively static conditions with limited number variables compared with a real situation, mainly bearing in mind the human body. The promising results in vitro do not guarantee their further efficacy in  vivo. For instance, in the design of nanocarriers that can target the tumor site and respond to the physiological signals in the tumor microenvironment, a complex scenario of requirements must be considered (long blood circulation time, ability to reach the target site, triggered release of payload, and biodegradability of the carrier). The precise integration of the responsiveness of a certain nanocarriers and the targeted physiological signal (e.g., pH, enzyme activity, or ROS) remains challenging. In order to improve the treatment efficacy, both the dynamic responsive behavior of the delivery system and detailed information on the targeted physiological signal, including distribution and concentration at the diseased site, should be thoroughly evaluated. Considering cancer applications, novel stimuli-responsive polymersomes with suitable targetability and stability with multistimuli-linkers that might be cleavable in extracellular and intracellular space in a stepwise manner might be promising. Moreover, 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 manufacturing process is always needed for translation to clinical use.

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