Polymersomes for Therapeutic Delivery of Protein

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Polymersomes for Therapeutic Delivery of Protein and Nucleic Acid Macromolecules: From Design to Therapeutic Applications Shoaib Iqbal, Mark Blenner, Angela Alexander-Bryant,* and Jessica Larsen*

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ABSTRACT: Macromolecule-based therapeutic agents, particularly proteins, antigens, monoclonal antibodies, transcription factors, nucleic acids, and gene editing enzymes, have the potential to oﬀer cures for previously untreatable diseases. However, they present an enormous delivery challenge due to poor absorption and rapid metabolism in the body. Polymersomes have tremendous potential in delivering these agents to their desired intracellular location due to increased circulation times, decreased macromolecule degradation, and decreased immune responses. In this Review, we highlight the key factors in design, development, and improved performance of these vesicles for macromolecular delivery. The recent progress made toward preclinical application of these vesicles for protein and gene delivery is also covered.

1. INTRODUCTION polymersomes are similar in morphology but are formed Macromolecular drugs such as peptides, proteins, antibodies, from amphiphilic block copolymers. They consist of an and nucleic acids are highly specific and potent therapeutic aqueous interior surrounded by a polymeric bilayer or agents. They have shown great clinical promise and represent a complicated interdigitated membrane structures, which are new paradigm of treatments for many diseases. As compared several folds thicker (up to 10 times) than their lipid ff counterparts, enabling mechanical and chemical stability and with small molecule drugs, these macromolecules o er 13 advantages such as higher disease specificity due to their decreasing leakiness of encapsulated payloads. Moreover, due 1 to the customizable and flexible design of copolymers, structural complexity and greater therapeutic activity. improved control over properties such as size, surface However, their structural complexity makes their therapeutic properties, functionalization, architecture, etc., and increased

Downloaded via CLEMSON UNIV on March 24, 2020 at 17:33:03 (UTC). delivery a challenging task. Macromolecular drugs are subject complexity in design, such as stimuli responsiveness, can be to poor in vivo stability due to physical and chemical achieved14,15 (Figure 1). Compared to liposomes, polymer- degradation, immune cell activation, short blood circulation somes oﬀer advantages due to higher molecular weight and half-lives, poor membrane permeability/cellular uptake, and ﬀ

See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. slower mobility of polymer chains, thereby o ering more lack of directed intracellular trafficking. To achieve high stringent supramolecular structures with highly entangled therapeutic efficacy using these macromolecules, there is an polymeric membranes.16 Compared to other polymeric increased interest in developing delivery strategies and nanovehicles such as nanoparticles,3 nanocapsules,17 and platforms. Remarkable progress has been made in the design nanogels,7 polymersomes offer the advantage of colloidal and fabrication of nanostructures for delivery of macro- stability and tunable membrane permeability.18 Given their molecular drugs, as they offer advantages including protection, design versatility compared to other polymeric carriers, they prolonged circulation, controlled release, and targeted delivery, − have also demonstrated successful encapsulation of macro- thereby improving safety and efficacy.2 8 molecules both inside the aqueous core as well as in the Intracellular organelles are natural vesicles involved in the − membranes, hence increasing the application potential.19 21 As organization, isolation, and protection of macromolecules during their functioning within cells. In an analogous way, compartmentalization by physical confinement of macro- Received: December 19, 2019 molecules in nanoscale dimensions can provide efficient Revised: February 20, 2020 transport of these fragile macromolecules in organelle-like Published: February 20, 2020 compartments.9 Polymeric vesicles termed “polymersomes” are synthetic analogues of these compartments and mimic cellular − structures and function.10 12 Like lipid-based vesicles,

a well-established nanoplatform, polymersomes are considered to be promising vehicles for biomedical applications. Recently, research on engineering polymersomes as platform for diagnosis and therapy has been summarized and highlighted − in several reviews.13,14,20,22 24 Du et al. reviewed recent advances in polymeric vesicles, including their preparation, stimuli-responsive behavior, and applications in medicine and beyond.13 They also summarized the modular potential of these vesicles for cancer theranostics.23 Kong and colleagues reviewed the improvements made for controlling size, shape, surface activity, and stimuli- responsiveness of polymersomes functions and their applications in diagnosis and therapy.24 Gu et al. highlighted the advances made in stimuli-responsive polymersomes and their potential biomedical applications.14 Zhong and colleagues briefly reviewed polymersomes as smart protein nanotherapeutics for targeted and efficient cancer therapy.2 However, a review of polymersomes for macromolecules has not received much attention in recent years. Here, we introduce some pioneering work in the field to describe the dynamics of polymersome assembly for macro- Figure 1. Schematic illustration of various advantages of polymer- molecule delivery. We will highlight the advances made to some-based delivery systems for macromolecule delivery. engineer polymersomes for efficient delivery of macromolecules, their therapeutic applications, and future potential for clinical translational.

Figure 2. Schematic demonstration of the self-assembly of block copolymers into polymersomes via two diﬀerent mechanisms, i.e. mechanism I and mechanism II.

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2. DESIGNING POLYMERSOMES FOR DELIVERY OF which can easily be accommodated inside the aqueous lumen MACROMOLECULES: DYNAMICS OF ASSEMBLY, of vesicles.20 PREPARATION, AND CONTROL OVER 2.2. Preparation Methods. The methods of preparing NANOPROPERTIES polymersomes for loading with macromolecules are mostly adapted from liposome preparation. They include film 2.1. Dynamics of Assembly. Amphiphilic block copoly- rehydration, direct hydration, solvent displacement, electro- mers contain adjacent blocks with different compositions, 19 poration, polymerization-induced self-assembly, double emul- solubility, and sequence distributions. They self-assemble in sification in a microfluidic device, and flash nanoprecipitation aqueous solution into supramolecular structures such as 19 (Table 1). For macromolecules, some of these methods cannot spherical micelles, cylindrical micelles, lamellae, and vesicles. be applied directly for scalable encapsulation and require Resultant architectures are dictated by several parameters such further optimization. as molecular weight, concentration, geometry, and block 2.2.1. Hydration of Polymer films. Film rehydration is a proportions of the polymers. Classically, the geometry of frequently applied method, where solutions of amphiphilic amphiphile assembly is described by packing parameter p=v/ block copolymers in organic solvents are evaporated to form alc (where v is the volume of densely packed hydrophobic thin films followed by rehydration or direct dissolution of 39 chain, a is the area of hydrophobic headgroup and lc is the polymeric materials. Control over polymersome size using 25 chain length of hydrophobic block). However, packing these methods is poor, producing a wide range of vesicle sizes, parameter p is based on geometric consideration and sometime thereby requiring postpreparation treatments, such as extrusion fails to describe the self-assembly of amphiphilic block via polycarbonate membrane, sonication, or freeze−thaw copolymers into various nanostructures.26 Therefore, block cycles.40,41 To improve the extrusion process following copolymers are preferably characterized by a synthetically conventional preparation methods, a hollow fiber (HF) fi accessible hydrophilic block fraction (f hydrophilic) rather than the membrane with a well-de ned pore size can be used for low- core packing parameter p. As a general rule of thumb, a pressure extrusion.42 This method doubled the encapsulation ± f hydrophilic of approximately 35 10% of an amphiphilic block of bovine hemoglobin (Hb) inside poly(butadiene-b-ethylene copolymer yields polymersomes.27 Other nanostructure oxide) (PBD-b-PEO) polymersomes compared to the manual morphologies such as micelles and worm-like micelles are extrusion method, allowing both improved encapsulation and − obtained at f hydrophilic > 0.50. large-scale preparation with lower applied pressures of 10 30 Self-assembly into vesicle structures is driven by noncovalent psi. However, the requirement of elevated temperatures and/or interactions of the hydrophobic blocks, i.e., hydrophobic effect, pressures limit the widespread application of this method. minimizing water interaction with hydrophobic chains. Hydro- A new method of “direct hydration” was proposed as a “ philic blocks favor the aqueous phase due to hydration forces hybrid of two preparation methods, namely, solvent ” “ ” as well as preventing contact of water with the hydrophobic dispersion and homopolymer addition , for encapsulating − block.28 30 Two mechanisms have been proposed for the biomacromolecules into high molecular weight block copolymers with rapid and increased encapsulation effi- polymersomes formation, commonly referred as mechanism I 43 and mechanism II31 (Figure 2). Mechanism I, bilayer-to-vesicle ciency. Blending poly(ethylene glycol) dimethyl ether (500 Da) with diblock poly(ethylene glycol)-b-poly(propylene model, is the widely accepted mechanism for polymersomes fi ° formation and begins by self-assembly of block copolymers sul de) (PEG-b-PPS) at 95 C for 20 min, followed by the addition of a protein solution, yielded polymersomes with high into spherical micelles followed by their evolution into bilayer ffi − fi sheets, and finally their closure into spherical vesicles.32 The encapsulation e ciency (15 37%) compared to lm rehydra- closure of bilayers into spherical vesicles is driven by edge tion. However, this method was only suitable for small-scale preparation (<1 mL) and thermostable proteins. To overcome energy which spontaneously bends the bilayer to lower fee the low encapsulation efficiency of macromolecules achieved energy. This transition has been experimentally observed in 33−35 by hydration methods, a general and novel method named several studies. In mechanism II, the spherical micelles “ ” ff progressive saturation was established and validated recently progressively grow in size due to solvent di usion thereby for maximal encapsulation of native protein by combining and lowering their bending energy by increasing the radius of optimizing various steps from multiple vesicle formation curvature, leading to polymersomes. This mechanism has been methods.44 Vesicles were formulated from PEO-b-PBD block predicted by simulation using the external potential dynamics 36 copolymers by iterative optimization. This method enabled (EPD) method. encapsulation of a variety of proteins ranging in molecular Polymersomes have been reported to successfully encapsu- weights from 17 to 450 kDa and increased encapsulation late a number of macromolecular payloads, such as proteins, ffi 20 e ciencies compared to conventional preparation methods of peptides, immunoglobulins, DNA, and siRNA. The hydro- film rehydration or direct hydration. philic nature of macromolecules allows them to be favorably 2.2.2. Cosolvent Addition. Cosolvent methods of for- encapsulated into the aqueous core or lumen of these vesicles mulation such as solvent displacement or nanoprecipitation where they are better protected from the external environment. begin by dropwise addition of amphiphilic block copolymer Polymersomes have also been shown to incorporate proteins in solution (in water-miscible solvent) into water under vigorous the bilayer membrane without loss of their conformation and stirring53 or vice versa.54 Although these methods achieve well- 37 resultant function. For plasmid DNA (pDNA), some sort of controlled polymersome size with narrow polydispersity, it condensation with polymers (polyplexes) is normally required requires removal of the organic solvent by evaporation, dialysis, before assembly into vesicles due to their large size and highly or freeze-drying before their downstream application, thereby charged nature.38 However, in the case of small interfering risking denaturation of fragile protein and DNA macro- RNA (siRNA) and antisense oligonucleotides (AON), molecules.57,58 Protection of costly payloads during freeze- condensation is usually not required due to their small size, drying can be mediated by the addition of lyoprotectants,

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although not all macromolecule activity will be maintained.59 A 52 41 47 49 54 , , , − , double emulsion solvent evaporation method was reported for 40 43 44 45 46 48 50 53 55 56 the development of bovine serum albumin (BSA) encapsulating PEG-b-poly(ε-caprolactone) PEG-b-PCL polymersomes.55 The obtained nanoparticles had high encapsulation efficiency (91%), which was attributed to strong interaction between the protein and the copolymer. 2.2.3. Electroporation. Electroporation has been widely used in molecular/cellular biology to introduce biomolecules e.g. pDNA into live cells. This method has been reported as a novel and convenient approach for efficient encapsulation of macromolecules into polymersomes by exploiting their supramolecular nature.45 Anionic biomacromolecules such as ciency, Advantages, and Limitations proteins,BSA,myoglobin(Mb),enzymes(Lysozyme), ffi

cult if water is used as the antisolvent antibodies (IgG), and nucleic acids (pDNA, siRNA) were ffi loaded into poly(2-(methacryloyloxy) ethyl phosphorylcho- line)-b-poly(2-(diisopropylamino)ethyl methacrylate) (PMPC- L/min), limitations on vesicle morphology and size μ b-PDPA)-based polymersomes by temporary destabilization of vesicle membranes. Due to the pH-sensitive nature of PMPC- b-PDPA (owing to pH-responsive nature of PDPA block), encapsulation of macromolecules was mediated by simply changing the pH to control self-assembly of the copolymer. Six different macromolecules were loaded into polymersomes by fl

m) electroporation without in uencing vesicle size and morphol- culty of solvent removal, risk of macromolecule denaturation, require the use of μ extrusion steps (few hours to overnight, higher temp and pressures) time ( additives (e.g., surfactants) hydrophilic cargos is much more di ﬃ ogy, indicating self-healing of the defects in polymersome high polydispersity, poor control over size, low yields of encapsulation, downstream only suitable for hydrophilic monomers (typically HPMA) low throughput production ( membranes after application of high voltage pulses. The surface charge was also reported to contribute to encapsulation eﬃciency, with higher loading for anionic biomacromolecules

450 kDa) prolonged preparation times (>40 h) compared with cationic molecules due to electrophoretic − motility-driven diﬀusion of anionic macromolecules into the inner core of polymersomes. This method was successful for encapsulating macromolecules (only charged proteins) in polymeric vesicles, though in general, polymersomes are somewhat resistant to electroporation.60 2.2.4. Bioinspired Approaches. An alternative to traditional 2990 kDa) favorable to encapsulation of only charged proteins, low yields of encapsulation

− methods for polymeric vesicles could be the use of membrane cient encapsulation risk of macromolecule denaturation

ﬃ proteins for assisting encapsulation. A phage transfection strategy was used for the loading of DNA into ABA-triblock copolymer vesicles.48 The membrane protein LamB, which demonstrated binding for lambda phages,61 was inserted into the polymersome membrane, thereby transferring DNA into the aqueous core. The study demonstrated both the functionality of proteins embedded in polymersome membranes and the protection of encapsulated DNA. Although not extensively evaluated for therapeutic application, a study investigated the incorporation of water channel protein cient encapsulation of hydrophilic macromolecules di macromolecules due to solvent-free nature reproducible and monodisperse vesicles, higher control over size subambient temp), facile loadingproduction of delicate macromolecules, scalable

ﬃ Aquaporin Z into symmetric poly-(2-methyloxazoline)-b- poly-(dimethylsiloxane)-b-poly-(2-methyloxazoline), PMOXA- 37 short preparation time (<1 h), maintain the bioactivity of macromolecules small scale (<1 mL), only suitable for thermostable macromolecules 19 facile loading of macromolecules membrane thickness mismatch, use of detergents, low yields of encapsulation, longer 52 self-assembled structures in nm range, mild reaction conditions (room or 95 e 43 scalable and rapid method of vesicles productionb-PDMS-b-PMOXA primarily limited to hydrophobic payloads, use of organicblock solvents, encapsulation of copolymer-based vesicles for trans- 15 straightforward, no special equipment, suitable for large number of 95 increased encapsulated quantity, suitability for various proteins (17 12 suitability for various macromolecules (13 − − − − − − − − 62 29 avoidance of postprocessing steps, complex nanostructures can be obtained, highly 40 16 >90 control over the vesicular structures, e port studies. Another study demonstrated the ion-selective permeability of polymersomes by inserting gramicidin biopores in PMOXA-b-PDMS-b-PMOXA-based vesicles membranes.63 Though only used for transport of small molecules, incorporating transport proteins in membranes could assist in designing and formulating polymeric vesicles with improved solvent displacement displacement displacement displacement

solvent-free 11 encapsulation and highly speciﬁc release of macromolecules. To allow loading of larger macromolecules into polymeric vesicles, a nuclear pore complex (which connects the cell cytoplasm and nucleus across the nuclear envelope) inspired system called nuclear envelope-like vesicle (NEV) was

method type EE (%) advantages49 limitations ref uidics solvent-free/ developed. A pH-responsive and photo cross-linkable diblock fl copolymer, PEO-b-poly[2-(diethylamino) ethyl methacrylate- self-assembly (PISA) lm rehydration solvent-free 5 ash nanoprecipitation solvent fi direct hydrationprogressive saturation solvent-free solvent-free 3 15 electroporation solvent-free 2 polymerization-induced bioinspirationmicro solvent-freesolvent injection 12 solvent emulsion phase transfer solvent fl Table 1. Preparation Methods of Protein and Nucleic Acid Macromolecules Loaded Polymersomes along with Their Encapsulation E stat-7-(2 methacryloyloxyethoxy)-4-methylcoumarin] [PEO-b-

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P(DEA-stat-CMA)], was designed and polymersomes were was reported recently.67 Using GOx during formulation formed in pure water for loading of sensitive and fragile allowed polymerization in normal air environment, while biomacromolecules (Hb, glucose oxidase (GOx) and FAM- retaining the activity of encapsulated protein (HRP). siRNA) by only changing the pH. Although the encapsulation Interestingly, the encapsulation efficiency of HRP and BSA efficiency was low (12−19%), the method offered a good was observed to be 50−52%. alternative for the loading of sensitive macromolecules with 2.2.7. Flash Nanoprecipitation. The assembly of block pH-responsive biomacromolecule delivery. This method has its copolymers into soft nanoarchitectures by high throughput own drawbacks. Incorporating a membrane protein into methods remains a challenge, and overcoming this problem vesicles membranes is energetically unfavored due to thickness will allow ease of fabrication, scalability, repeatability and high mismatch between hydrophobic domain of the membrane loading efficiency critical for clinical translation. To address protein and the vesicle membrane,64 and requirement of this, a recently developed flash nanoprecipitation (FNP) detergent for water insoluble proteins which affect the method has been reported as a scalable and rapid technique structure of the membrane. for formulating vesicular nanoarchitectures.56 A range of 2.2.5. Microfluidics. Microfluidic devices provide a higher nanostructures, such as filomicelles, nanospheres, tubular, degree of control over self-assembly in a locally modulated and multilamellar vesicles were obtained from block copoly- environment, as exists in cellular microenvironments.65 mers PEG-b-PPS. The size and polydispersity of formed Complex multicompartment polymersomes structures have polymersomes was decreased by several impingements, also been obtained by using a simple coflow microfluidic allowing fabrication of monodisperse polymersomes on a device.52 In an effort to develop a more robust production gram scale within minutes. This methodology was suitable for method, a flow-focusing microfluidic device was developed loading of both hydrophilic and hydrophobic molecules into which provided more favorable conditions for encapsulation, the aqueous core/lumen and membrane of the polymersomes, thereby enhancing encapsulation efficiency.50 By changing the respectively. The encapsulated enzyme (alkaline phosphatase, pH of the flows within the microchannels, self-assembly of AP) retained its activity after release from the lysed PMPC-b-PDPA block copolymer was induced. The encapsu- polymersomes. lation efficiency of proteins by this method (29%) was Given the sensitivity of macromolecules to the manufactur- equivalent to the standard aqueous polymersome production ing process (such as organic solvent, temperature, pH, methods (27%), but it eliminated the use of organic solvent oxidation etc.), it is necessary to characterize them for and batch to batch variations in polymersome preparation. structure (secondary/tertiary), size, location inside the vesicles, Although microfluidics along with film hydration has proven to and function for successful delivery. The choice of character- be most amenable for loading of protein macromolecules due ization technique can be exclusive for the study of particular to removal of the sonication postprocessing step and avoidance properties, or they can be combined to allow complete of organic solvents, thereby retaining protein conformation and characterization. Microscopy-based techniques, e.g. cryo trans- activity, the associated drawback of this platform is low mission electron microscopy (TEM),68 atomic force micros- throughput production (μL/min) and limitations on vesicle copy (AFM),62 fluorescence microscopy,40 and confocal laser morphology and size (microscale) due to channel dimensions. scanning microscopy (CLSM),69 have been used to provide The use of microfluidics for polymersome assembly of information on the size, morphology, structure, location, and poly(ethylene glycol)-b-poly(D,L-lactic acid) copolymers loading of macromolecules in the vesicles. Gel characterization (PEG-b-PLA) has also been effective in encapsulating such as SDS-PAGE has been carried out to confirm the biological machinery needed for expression of proteins by encapsulation of macromolecules within vesicles.70 Spectro- water-in-oil-in-water double-emulsion drops.51 MreB, a mem- scopic technique such as UV−vis have been used to verify brane-related bacterial protein, was expressed in a cell-free functional capacity/biological activity of macromolecules after polymersome expression system in high yields within a few encapsulation71 and tertiary structures.72 Fluorescence corre- hours, with most of the protein suspended inside the interior of lation spectroscopy (FCS) has been applied to quantitatively vesicles. Protein release from vesicles was triggered by osmotic differentiate between free against object associated fluores- shock, owing to a semipermeable membrane. cence based on differences in diffusion coefficients.73 Influence 2.2.6. Polymerization-Induced Self-Assembly (PISA). Con- of formulation process on protein secondary structure has been ventional self-assembly methods often require organic solvents, measured by Circular dichroism (CD).74 Fourier transform making them incompatible for certain proteins. Additionally, infrared (FTIR) has also been employed for loading of due to low concentrations as well as multiple formulation and macromolecules into vesicles,55 while fluorescence spectrosco- purification steps, their potential scalability is limited. Several py for the encapsulation quantification.47 Differential scanning strategies have been devised recently to overcome this calorimetry (DSC) has also been reported to confirm the limitation. PISA has offered an efficient way to encapsulate physical interaction between copolymers and cargos upon functional macromolecules (e.g., BSA) with large-scale loading.55 Quantitative characterization such as oxygen production potential by utilizing the living polymerization of carrying capacity of Hb75 and fluorescent shift measurements a solvophobic polymer to drive self-assembly in situ.46,47 An of substrate by encapsulated and native enzymes (granzyme B, initiator-free visible light-mediated PISA (photo-PISA) using GrB)76 have been reported to ensure the bioactivity of the commercial monomer and under mild, aqueous conditions was macromolecule payloads after encapsulation into the polymer- reported for the loading of macromolecules such as horseradish somes. peroxidase (HRP) and GOx.66 The approach demonstrated In summary, preparation and loading techniques have been the functionality of the encapsulated proteins. However, this developed that could allow for enhanced macromolecule method is only suitable for hydrophilic monomers which form delivery. Key criteria in chosen preparation and loading hydrophobic polymers. An efficient enzyme assisted photo- techniques that will ease translation to clinical applications PISA platform that was compatible with open air environments include narrow polydispersity, higher encapsulation efficien-

E https://dx.doi.org/10.1021/acs.biomac.9b01754 Biomacromolecules XXXX, XXX, XXX−XXX Biomacromolecules pubs.acs.org/Biomac Review cies, maintenance of macromolecule structure and bioactivities rate and extent of permeability, as well as enhanced stability upon encapsulation, and batch scale production leading to with preservation of size and structure under physiological large enough doses for therapeutic efficacy. As a field, several conditions even after freeze-drying and centrifugation treat- techniques have been developed to enhance encapsulation ment. A polyelectrolyte-based diblock copolymer of poly- efficiencies while maintaining narrow polydispersity. However, (styrene)-b-poly(acrylic acid), PS-b-PAA, easily formed poly- it is clear that more work needs to be done to ensure mersome aggregates through the addition of cationic proteins maintenance of macromolecule function and activity, as well as and peptides to the copolymer.86 Vesicle formation occurred scaling up production of polymersomes to therapeutically due to electrostatic interactions between the cationic proteins relevant doses. (cytochrome c (CC), green fluorescent protein (GFP), and 2.3. Nanoproperties: Influence of Polymer Composi- Acropora millepora derived GFP, amGFP), and anionic PAA in tion, Architecture, Membrane Features, Polymersomes the polymer blocks which led to entrapment of proteins in the Shape and Stimuli Responsiveness. The control over membranes. Higher encapsulation efficiencies of 35 and 66% nanoscale properties of polymeric vesicles plays a critical role were observed for CC and amGFP respectively. Vesicles were in their applications in nanomedicine. These nanoproperties only observed when using cationic proteins, whereas micelles dictate the physicochemical characteristics, surface features, were formed using anionic proteins. Conjugation of polymers mechanical stability, and pattern of drug release. The size of with proteins termed “polymer-protein conjugates” have been polymeric nanocarriers affects their in vivo fate through shown to self-aggregate into submicron vesicles.87 An interactions with serum proteins,77 biodistribution,78,79 and amphiphilic protein−polymer construct formed by conjugation pharmacokinetics.80 Polymersomes can be generated with a of BSA with poly(N-isopropylacrylamide) (PNIPAM) (BSA-b- range of different sizes from tens to thousands of nanome- PNIPAM) exhibited temperature responsiveness and displayed ters15,27 Their size is influenced by various macromolecular self-assembly behavior at water/oil interfaces to form semi- (thermodynamics) parameters, such as the composition of the permeable compartments called “proteinosomes”.74 The polymeric materials, polymer concentration, and fabrication micrometer sized vesicles (50−150 μm) were able to method, including self-assembly, extrusion, and freeze−thaw encapsulate a range of macromolecules, such as Mb, ferritin, − cycles.81 84 Impact of physicochemical properties of polymer- RNA polymerases, lipoprotein lipase (LPL), and AP. The somes on macromolecule loadings are summarized in Table 2. proteinosomes also assisted in gene-directed protein synthesis. Rod−coil diblock copolymers such as polystyrene-b-poly(L- Table 2. Impact of Physicochemical Properties of isocyanoalanine(2-thiophen-3-yl-ethyl)amide) (PS-b-PIAT) Polymersomes on Macromolecule Loadings which consist of a flexible polystyrene tail and a rigid polyisocyanide block have been reported to form porous property impact on macromolecule cargos ref vesicles for encapsulation of macromolecules such as Candida polymer higher loadings of macromolecules (by ionic 75, 86, 88 antarctica Lipase B (CAL B) enzyme.107 Using this rod−coil (composition blocks, by heterotriblocks) diblock copolymer, a two-enzyme-based system was con- and topology) reduced/controlled release of macromolecules 70, 89 (by graft copolymers, by ionic polymer blocks) structed with positional control over their location inside the positional control over macromolecule 90−92 polymersomes which was achieved by using lyophilization encapsulation during the formulation of polymersomes.90 By mixing an shape transport across cellular barriers 93−95 anchor block copolymer (to allow surface functionalization) membrane reduced/controlled release of macromolecules 96−99 with PS-b-PIAT copolymer, polymersomes with surface (by increasing Mw of hydrophobic block, i.e. 91 thickness, by cross-linking of hydrophobic attached macromolecules were also formulated. Antibody- blocks, by doing diblock with a triblock functionalized magnetic polymersomes were also prepared by polymer) blending poly(trimethylene carbonate)-b-poly(glutamic acid) higher loadings of macromolecules (by 100,101 asymmetric membrane, by charged membranes) block copolymer (PTMC-b-PGA) with a maleimide end- − capped copolymer to allow surface functionalization with stimuli- controlled release of macromolecules (by pH, 102 106 108 responsive oxidation, UV, sugar sensitive etc.) Trastuzumab antibody. Monitoring the position of the payload is an important consideration in designing vesicles for 2.3.1. Polymer Composition and Topology. As the building macromolecule delivery. To this, spatial control over the block of the polymersomes, copolymers form well-defined encapsulant and identifying the precise location inside structures both in solution and in solid state. However, their polymersomes has been addressed by synthesizing a phase behavior is significantly changed when additional energy protein−polymer bioconjugate, PNIPAM-b-amilFP497.92 The contributions from electrostatic interactions are considered. conjugate was composed of thermoresponsive poly(N- Formation of polymeric vesicles with polyion complex isopropylacrylamide) (PNIPAM) and a variant of green membranes termed “PICosomes” has been achieved by fluorescent protein (amilFP497). Above 37 °C, this bio- complexing oppositely charged block polymers, called conjugate formed vesicles, and by using the Forster resonance ionomers, with biofunctional macromolecules.69,85 The energy transfer along with fluorescence lifetime imaging oppositely charged polyelectrolytes with PEG-poly(α,β- microscopy (FRET-FLIM), the location of biomolecules, i.e., aspartic acid) as an anionomer and PEG-poly([2-aminoethyl]- light-harvesting protein phycoerythrin 545 (PE545), and α,β-aspartamide) as a cationomer, along with the addition of a doxorubicin inside the polymersomes were studied. The macromolecule (such as protein or nucleic acid), afforded PE545 was found to be encapsulated primarily within the micrometer-sized vesicles in aqueous solution. By simply membrane. changing the total polymer concentration of these ionomers, Polymer topology plays an important role in the perform- the size of PICosomes was controlled to sub micrometer ance of the resultant polymersomes. Compared to block diameters, i.e., 100 to 400 nm.73 Moreover, cross-linking the copolymers, polymersomes formed from biodegradable membrane of these PICosomes allowed fine control over the amphiphilic graft copolymers, i.e., poly(lactide-co-diazido-

F https://dx.doi.org/10.1021/acs.biomac.9b01754 Biomacromolecules XXXX, XXX, XXX−XXX Biomacromolecules pubs.acs.org/Biomac Review methyl trimethylene carbonate)-g-PEG (P(LA-co-DAC)-g- Under mild formulation conditions the strategy offered 100% PEG), demonstrated improved resistance to protein adsorp- encapsulation of enzymes while retention of activity. 70 tion and a reduced rate of release of encapsulated protein. Biodegradable PEG-b-poly-(D,L-lactide) (PEG-b-PDLLA)- Such modification was beneficial in prolonging blood based tubular polymersomes were formulated by spontaneous circulation time and sustaining delivery of macromolecules. elongation of spherical polymersomes.95 The size was In a systematic investigation, an amphiphilic heterotriblock controlled osmotically using dialysis. Tethering of functional copolymer PEG-b-(poly(ε-caprolactone-co-lactic acid)-b-PEG proteins (enhanced GFP) on their surface offered potential (PEG-b-P(CL-co-LA)-b-PEG) was designed and synthesized application of these vesicles for biomedical research. We have for encapsulating Hb.75 The polymersomes formed from these recently reported the prolate-like polymersomes for efficient asymmetric blocks achieved enhanced encapsulation efficiency delivery of hydrophilic and hydrophobic cargos.93 PEG-b-PLA compared to diblock counterparts due to avoidance of long- polymersomes with prolate morphology showed a higher chain PEG stretching toward the internal cavity in the triblock encapsulation efficiency of Fluorescein-BSA (23%) compared copolymer, leading to an increased volume for protein loading. to traditional spheres (13%). Increased cellular uptake of the ABC triblock polymers (with A and C as hydrophilic but nonspherical polymersomes was observed in SH-SY5Y cells different composition) have been reported to form polymer- which was attributed to greater contact between their flattened − somes with asymmetric membranes.109 112 Biodegradable shape and cell surface. “chimeric polymersomes” formulated from an asymmetric 2.3.3. Membrane Properties. Properties of vesicle mem- triblock copolymer, PEG-b-PCL-b-poly(diethyl-amino) ethyl branes like fluidity and thickness depend on components such methacrylate(PEG-b-PCL-b-PDEA, which contain a distinct as the number of block units in the amphiphilic copolymer and interior separated from the outside by an asymmetric the molecular weight of the hydrophobic block.123,124 The membrane, was prepared for cytosolic delivery of proteins, membrane fluidity has been shown to decrease with increasing such as BSA, CC, lysozyme (Lys), ovalbumin (OVA), and molecular weight, becoming more pronounced with longer immunoglobulin G (IgG).88 The loading efficiency was higher hydrophobic blocks due to chain entanglement. The thickness than typically observed in polymersomes, with some proteins of the membrane of the polymersome can also be manipulated exhibiting up to 99% loading efficiency. This was attributed to to explore its effect on controlled release of payloads. The effective electrostatic interactions and hydrogen bonding ability of polymersome bilayer thickness to regulate gaseous between PDEA blocks and proteins. Cationic polymersomes ligand binding and release kinetics of encapsulated Hb has were formulated from three hydrophilic and biocompatible been studied by formulating vesicles from polymers of varied cationic random copolymers polymerized from N,N- molecular weights, spanning from 1.8 to 10.4 kDa.96 A (dimethylamino)ethyl methacrylate (DMAEM) and different hydrophobic membrane thickness ranging from 3 to 13 nm molar ratios of mPEG.89 In these vesicles, the membrane regulated oxygen offloading and delayed nitric oxide binding to consisted of PEG and the cationic DMAEM block formed the encapsulated Hb at increased membrane thickness, demon- core and surface, rendering a positive charge to these vesicles. strating the potential use of these polymersomes in transfusion At acidic pH, vesicles transformed into micelles, releasing their medicine as a Hb-based oxygen carrier. A unique polymersome hydrophilic payloads. In the presence of macromolecules, such differing from conventional polymersomes in terms of its as human serum albumin (HSA) and pDNA, polymersomes bilayer membrane was formulated by two different amphiphilic retained their stability, demonstrating their potential for diblock copolymers, PEG-b-PCL and dextran-b-PCL (DEX-b- sustained delivery applications. Multicompartmentalized poly- PCL), in an asymmetric “phase-guided assembly”.100 Proteins mersomes have also been investigated for encapsulation of were loaded with high efficiency (89%) due to the macromolecules.113 A combination of di- and triblock thermodynamically favored partition and asymmetric bilayer, copolymers, i.e. PS-b-PIAT and PMOXA-b-PDMS-b-PMOXA with encapsulated erythropoietin (EPO) retaining well- forming vesicles by film hydration followed by direct preserved bioactivity observed in a UT-7 cell proliferation dissolution resulted in selective encapsulation of macro- assay. The system design offered the possibility of cross- molecules, including GFP and Cy5-IgG. Both macromolecules linking, thereby enhancing mechanical strength. were encapsulated in separate compartments mimicking Permeability of membranes has been tuned by cross-linking cellular compartmentalization as an “organelle mimic”. throughout the bilayer membrane to endow structural 2.3.2. Polymersome Shape. In addition to size, surface robustness and encapsulation stability to hydrophilic payloads. charge, and material composition, nanoparticles shape has also Polymersomes with self-immolative side linkages in the been reported to affect their transport across various barriers hydrophobic block underwent intracellular stimuli triggered inside the cell.114 The nonspherical shape of the polymersomes reactions, resulting in concurrent cross-linking and hydro- mimics cells and organelles that we find in nature to facilitate phobic to hydrophilic transition of the bilayer membranes.97 In biochemical processes.83,115,116 Similar to inorganic nano- a similar fashion, a light induced cross-linking and hydrophobic particles where nonspherical shaped particles are routinely to hydrophilic transition was achieved for sustained release of synthesized and used in various applications, recent years have cargos.98,125 A glutathione triggered cleavage of camptothecin witnessed an increased interest in the development of in polyprodrug-gated cross-linked vesicles (GCVs) resulted in − nonspherical polymersomes.117 119 Polymersomes with a generation of hydrophilic mesh channels in the bilayer bowl-shaped structure referred to as “stomatocytes” have membranes and permeabilization of the whole vesicle been recently formulated from PEG-b-PS spherical polymer- structure.126 These membrane permeabilization strategies somes.120,121 Natural penetrating peptide functionalized allowedsynchronizedreleaseofthecargos.Membrane stomatocytes displayed rapid and effective penetration their permeability has also been tweaked for selective transport of use as efficient delivery carrier.122 Sensitive macromolecules ions by inserting biopores in polymersome membranes.63 Such such as enzymes and GOx and catalase were also loaded with engineering enables applications in nanoreactors or artificial high encapsulation efficiency into PEG-b-PS stomatocytes.94 organelles. Biodegradable and membrane ionizable polymer-

G https://dx.doi.org/10.1021/acs.biomac.9b01754 Biomacromolecules XXXX, XXX, XXX−XXX Biomacromolecules Table 3. Polymersome-Based Vesicles for Delivery of Protein, Peptides, Immunoglobulin, and Polysaccharide Macromolecules

polymer fabrication method protein size remarks ref A. polymersomes with nonbiodegradable hydrophobic blocks PEO-b-PEE or PEO-b-PBD electroporation, film rehydration, bulk Mb, Hb, Fluor-albumin nm−μm provides some indication of in vitro compatibility and 40 rehydration stability in plasma PEO-b-PBD film rehydration Hb <200 nm influence of polymer mol. wt. on physical properties and 41 oxygen binding capabilities were studied PEO-b-PBD aq. self-assembly and hollow fiber Hb 80−90 nm scale-up procedure for homogeneous vesicle preparations 42 extrusion method PEO-b-PBD film rehydration TNFα 157 nm surface functionalization for targeted delivery of vesicles 138 PEG-b-PBOx reversed solvent injection insulin 114−387 nm sugar responsive delivery vehicles 106 PEO-b-PBD film rehydration Hb 380−417 nm influence of different hydrophobic membrane thicknesses 96 on exchange of gases PWO-b-PBD progressive saturation Mb, Hb, BSA, IgG, catalase, 130−200 nm achieved improved encapsulation of functional protein in 44 fibrinogen, apoferritin vesicles PMOXA-b-PDMS-b-PMOXA and PS-b-PIAT film rehydration followed by direct GFP, Cy5-IgG 160 nm multicompartmentalized polymersomes for selective 113 dissolution encapsulation PMOXA-b-PDMS-b-PMOXA film rehydration BSA, NGF, CD109 65−75 nm macromolecule vesicles for therapy of neurological 139 disorders B. polymersomes with biodegradable polyesters as hydrophobic blocks PLA-b-F127-b-PLA solvent injection insulin 56 nm in vitro and in vivo release behavior and application as oral 140 delivery carrier for insulin were investigated PEO-b-PCL-b-PAA direct dissolution of copolymer BSA 242−398 nm demonstrated formation of biodegradable and fully 68 biocompatible polymeric vesicles pubs.acs.org/Biomac H PEG-b-PCL and PEG-b-PLA-b-PEG double emulsion solvent evaporation Hb 200 nm guide for tailored design of polymersomes for blood 133 substitutes PEO-b-PCL and PEO-b-PLA film rehydration human and bovine Hb 100−130 nm development of biodegradable and biocompatible Hb 134 carrier PEG-b-PCL-b-PDEA film rehydration BSA, CC, lysozyme, OVA, 130−175 nm chimaeric polymersomes for delivery of exogenous 88 IgG proteins to cancer cells PEG-b-PCL and DEX-b-PCL phase-guided assembly FITC-DEX, erythropoietin 2−9 μm efficient encapsulation of proteins due to thermodynamic 100 partitioning PEAG-g-PCL film rehydration and lyophilization− Hb 142 nm potential artificial oxygen and carriers 71 rehydration Gal-PEG-b-PCL PEG-b-PCL-b-PDEA PEG-b-PCL solubilizing polymers followed by protein CC and GrB 95−200 nm multifunctional platform effectively caused apoptosis of 141 addition HepG2 cells PEG-b-PLGA reversed solvent injection BSA-Gd 280 nm theranostic vesicles for in vitro and in vivo MRI 142 PEG-b-P(CL-co-LA)-b-PEG modified film hydration Hb 390 nm better therapeutic performance in a rat model of acute 75 https://dx.doi.org/10.1021/acs.biomac.9b01754 anemia β Biomacromolecules PEG-b-PLA NHS-PEG-NHS and ApoE solvent injection -galactosidase 411 nm controlled delivery of macromolecules for enzyme therapy 58 PEG-b-PCL double emulsification BSA 78 nm potential nanocarrier for delivery of macromolecules 55 PCL-b-PEG-b-PCL, DOTAP, DSPE-PEG-Mannose double emulsion method (w/o/w) OVA 234 nm lipid-hybrid polymersomes for targeted delivery of antigen 72 and adjuvant P(LA-co-DAC)-g-PEG reversed solvent injection Hb ≈ 100 nm grafted polymer vesicles exhibited stable membrane 70

XX X,XXX XXX, XXXX, packing and slowed release rate PAA-ONB-PMCL solvent injection eGFP 80 nm demonstrated phototriggerable nanovesicles for stimuli- 104 responsive delivery Review − XXX Biomacromolecules Table 3. continued

polymer fabrication method protein size remarks ref C. polymersomes with biodegradable polycarbonates as hydrophobic blocks − PEG-b-PTMC (with Ac, COOH, NH2 modifications) dispersing polymer in buffer at room FITC-BSA, FITC-CC, CC 94 112 nm membrane ionizable biodegradable vesicles enhanced 101 temperature apoptosis in MCF-7 cells PGA-b-PTMA. PEG-b-PGA-b-PTMC solvent assisted dispersion trastuzumab 165 nm antibody functionalized polymersomes for bone imaging 108 PEG-b-P(DTC-TMC)-b-PEI and ApoE-PEG-b-P(DTC- solvent injection FITC-CC, saporin 80−86 nm targeted and stimuli-responsive vesicles established 143 TMC) efficiency in glioblastoma therapy PEG-b-P(DTC-TMC)-b-PEI and ANG-PEG-b-P(DTC- solvent injection FITC-CC, saporin 76 nm targeted and stimuli-responsive vesicles established 144 TMC) efficiency in glioblastoma therapy PEG-b-P(TMC-DTC)-b-PEI and CPP33-PEG-b-P(TMC- solvent exchange method FITC-CC, GrB 82−90 nm targeted and stimuli-responsive vesicles established 76 DTC) efficiency in lung cancer therapy PEG-b-P(TMC-co-DTC)-spermine, cRGD-PEG-b-P(TMC- solvent exchange method Cy5-CC, FITC-CC 65 nm dual functionalized vesicles for cytosolic delivery of 145 co-DTC), Mal-PEG-b-P(TMC-co-DTC) and GALA macromolecules Acupa-PEG-b-PTMBPEC-b-PSAC PEG-b-PTMBPEC-b- solvent injection BSA, CC, GrB 157−175 nm efficient delivery of proteins into prostate cancer (LNCaP 146 PSAC cells) and improved pharmacokinetics in vivo PEG-b-P(TMC-DTC)-b-SP, Mal-PEG-b-P(TMC-DTC) solvent injection Cy5-CC, GrB 90 nm targeted protein therapy for multiple myeloma 147 D. polymersomes with poly(propylene sulfide) as hydrophobic blocks PEG-b-PPS direct hydration OVA, BSA, r-globulin, ≈ 500 nm new method for vesicle assembly 43 proteinase K PEG-b-PPS film rehydration OVA ≈ 160 nm oxidation-sensitive vesicles as a vaccine delivery platform 148 PEG-b-PPS film rehydration OVA 125 nm enhanced antigen specific response in vivo 149 − PEG-b-PPS film rehydration HIV-1 Gag Ag85Bp25 148 155 nm development of vesicle-based novel pediatric vaccines 150 pubs.acs.org/Biomac − I PEG-b-PPS flash nanoprecipitation DEX, GFP, BSA, OVA, AP 70 116 nm provided new route for high throughput nanofabrication 56

PEG-b-PPS along with ethyl eosin film rehydration OVA MHC I epitope ≈ 254 nm demonstrated an optofluidic method for precisely 105 SIINFEKL controlled rupture and intracellular delivery E. polymersomes with methacrylate-based hydrophobic blocks PMPC-b-PDPA microfluidic device BSA 75−275 nm robust and pH induced self-assembly production process 50 PMPC-b-PDPA film rehydration AF546-IgG, Ag-IgG ≈ 200 nm intracellular delivery of antibodies 151 PEG-b-PDEA adjusting pH of polymer and protein to FITC-BSA, FITC-CC 55−67 nm dual (pH and reduction) responsive vesicles enhanced 152 neutral apoptosis of MCF-7 cells PMPC-b-PDPA electroporation BSA, Mb, IgG, pDNA, ≈ 200 nm convenient and broader application of the approach 45 siRNA, Lz PMPC-b-PDPA film rehydration AF546-IgG, NF-κB-p65 Ab N.A. demonstrated delivery of large antibodies by vesicles 153 PEO-b-P(DEA-stat-CMA) reversed solvent injection GOx, Hb, FAM-siRNA 100 nm encapsulated macromolecule vesicles were formed directly 49 in water PEG-b-PAA(SH)-b-PDEA adjusting the pH of polymer and protein FITC-BSA, FITC-CC, CC 35 nm dual (pH and reduction) responsive vesicles enhanced 154 https://dx.doi.org/10.1021/acs.biomac.9b01754 aqueous solution at pH 6.0−8.0 apoptosis of MCF-7, HeLa and 293T cells − Biomacromolecules Anis-PEG-b-PTTMA-b-PAA and PEG-b-PTTMA-b-PAA solvent injection FITC-CC, GrB 152 171 nm highly potent and specific vesicles for lung cancers (H460 155 cells) PEG-b-P(HPMA-LA)-b-PAA and Anis-PEG-b-P(HPMALA)- solvent injection FITC-CC, Cy5-CC, GrB 167 nm demonstrated efficient inhibition of H460 lung tumor in 156 b-PAA mice PEG-b-PHPMA photo-PISA BSA 200−400 nm photo-PISA as a facile and rapid method for protein 46 encapsulation while retaining the activity XX X,XXX XXX, XXXX, PGMA-b-PHPMA PISA BSA 350 nm in situ loading of delicate macromolecules by using PISA 47 PEG-b-PHPMA photo-PISA GFP, HRP, GOx ≈ 250 nm PISA-based vesicles with permeable PHPMA membrane 66 was investigated Review PGMA-b-PHPMA enzyme assisted photo-PISA HRP, BSA ≈ 500 nm a robust oxygen-tolerant method to prepare 67 − XXX PEO-b-PTTAMA reversed solvent injection AP ≈ 680 nm pH responsive release of macromolecules 102 Biomacromolecules Table 3. continued

polymer fabrication method protein size remarks ref F. polymersomes with miscellaneous polymeric blocks PEG-b-P(Asp) and PEG-b-P(Asp-AP) aq. solution of oppositely charged block FITC-DEX 10 μm novel polymer vesicles called PICsome and their biological 69 ionomers and protein relevance was established PEG-b-P(Asp) and PEG-b-P(Asp-AP) aq. solution of oppositely charged block Mb 0.5−5 μm novel polymer vesicles called PICsome and their biological 85 ionomers and protein relevance was established PEG-b-P(Asp), PEG-b-P(Asp-AP) and Homo-P(Asp-AP) aq. solution of oppositely charged block FITC-DEX 100−400 nm size was controlled by changing the polymer concentration 73 ionomers and protein and vesicles increased circulation in vivo PEG-b-PAA-b-PNIPAM aq. self-assembly in water via increasing FITC-DEX 180 nm reversibly cross-linked and temperature-responsive 157 temperature polymersomes for triggered protein release PS-b-PIAT and PS-b-PEG-TAT solvent injection GFP, HRP 114 nm reported intracellular catalysis and functionality as artificial 158 organelles PS-b-PAA solvent injection CC, amGFP, poly-L-lysine 200−400 nm used polyelectrolyte-based diblock copolymers, overcome 86 problems with traditional encapsulation PEG-b-PAA-b-PNIPAM aq. dissolution of polymer and protein CC 150−170 nm dual (reduction and temperature) responsive vesicles with 159 intracellular delivery in MCF-7 cells PNIPAAm-BSA PNIPAAm-Mb PNIPAAm-Hb double emulsification peGFP and no. of 50−150 μm proteinosomes for in vitro gene expression 74 macromolecules PNIPAM-b-amilFP497 heating at 37 °C and self-assembly PE545 1.7 μm efficient identification of location of macromolecules 92 within vesicles PEG-poly-Ser-Ketal solvent injection Insulin, GOx, bovine 324 nm glucose responsive vesicles for therapeutic delivery of 160 Catalase insulin

PLys-g-PLys(AA) solvent injection VEGF 264 nm controlled delivery of macromolecules for regenerative 161 pubs.acs.org/Biomac

J medicine

PEG-b-PAPA-b-PAsp and cRGD-CLP solvent exchange method FITC-CC, saporin 81 to 86 nm lipopepsomes as nanocarriers for protein therapy of lung 162 tumors in mice https://dx.doi.org/10.1021/acs.biomac.9b01754 Biomacromolecules XX X,XXX XXX, XXXX, Review − XXX Biomacromolecules pubs.acs.org/Biomac Review somes were prepared from PEG-b-poly(trimethylene carbo- grated into micellar-like structures, which resulted in release of nate) (PEG-b-PTMC) block copolymers with acrylate, their macromolecular eGFP payloads. An optical approach was carboxylic acid, and amine groups along the PTMC block for investigated for delivery of oxidation-sensitive polymersomes efficient intracellular protein delivery.101 The charged mem- into cells.105 By using the photosensitizer ethyl eosin, which brane facilitated efficient loading and stabilization of proteins associated with vesicle membranes, the hydrophilicity of the via electrostatic interactions. Furthermore, an accelerated hydrophobic block was oxidatively increased under optical protein release was observed at endosomal pH due to excitation. This optofluidic interaction led to transition to weakened protein−membrane interactions. A simple method smaller vesicles and micelles along with rupture of vesicles and of regulating the membrane porosity has been reported by payload release. This platform mediated delivery of peptide preparing polymersomes with a diblock copolymer PEG-b- antigens to dendritic cells and payload release was found to PBD, and the triblock copolymer PEG-b-poly(propylene occur within a few seconds of illumination. A smart sugar- oxide)-b-PEG (PEG-b-PPO-b-PEG).99 The diblock-based responsive polymersome was developed from boronic acid- vesicles were found to be nonporous while vesicles doped containing block copolymers, consisting of poly(styrene with 25 mol % PEG-b-PPO-b-PEG improved permeability to boroxole) (PBOx) and PEG-b-PBOx.106 The encapsulated molecules less than 5 kDa. Macromolecules with a size of >10 FITC-insulin was released only in the presence of sugars at kDa were retained within the aqueous cores of these vesicles physiologically relevant pH due to monosaccharide-triggered after encapsulation. Incorporation of lipids in polymersomes disassembly of vesicles in aqueous solutions at neutral pH. In membranes has been reported to formulate hybrid mem- subsequent studies, the sensitivity of the polymer was increased branes.127 By formulating hybrid giant PDMS-g-PEO-based by formulating polymersomes from sequence-specific copoly- unilamellar vesicles with zwitterionic phospholipids DPPC: mer of styrene boroxole and N-functionalized maleimide.130 1,2-dipalmitoyl-sn-glycero-3 phosphocholine, and POPC: The presence of hydrophilic solubilizing groups between the palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), hybrid mem- boroxole moiety allowed polymersome disassembly at lower branes were generated. Such hybrid membranes due to glucose levels. presence of polymersomes and liposomes features are potentially interesting as they allow incorporation of proteins 3. POLYMERSOMES FOR THERAPEUTIC PROTEIN into the membranes.128 DELIVERY 2.3.4. Stimuli Responsiveness. Efficient targeting and better Proteins are a unique class of macromolecules with both high control over release mechanisms of nanocarriers allow specificities and activities.131 Many proteins have demonstrated enhanced therapeutic efficacy by preventing nonspecific cell/ significant therapeutic activity and thus have emerged as tissue distribution and premature release. Tremendous advanced alternatives to conventional small molecule ther- progress has been recently to develop stimuli responsive apeutics. However, their therapeutic functions are severely nanosystems often called as “smart nanocarriers”. Polymer- limited by their short in vivo half-lives and inability to somes have also seen significant advancements to their independently overcome extra- and intracellular barriers.132 responsiveness against external stimuli such as pH, temper- The development of nanocarriers has demonstrated prelimi- ature, oxidation, reduction, and light. Recently new stimuli nary success in overcoming these barriers to protein delivery,2,5 responsive approaches such as electric field, magnetic, sugar, and are thus a potential therapeutic alternative to native and ultrasound have also been used for formulating polymer- proteins. somes. As these smart polymersomes have been extensively Encapsulation of protein into polymersomes has been reviewed recently,14,22,129 here we will focus on the impact of proven to be a successful method of improving half-life and stimuli responsiveness on macromolecule delivery. Specific overcoming physiological barriers while retaining activity.20 examples of single and dual responsive polymersomes with The polymersome-based vesicles that have been used for respect to therapeutic applications are highlighted in Sections 3 delivery of protein macromolecules are listed in Table 3. Initial and 4. work by Discher and colleagues demonstrated the possibility of Polymersomes formulated from amphiphilic diblock copoly- encapsulating proteins (Mb, Hb, and albumin) into polymer- mer, PEO-b-poly(2-((((5-methyl-2-(2,4,6-trimethoxyphenyl)- somes.40 As an analogue of natural erythrocytes, these self- 1,3-dioxan-5-yl)methoxy)carbonyl)amino)ethyl methacrylate) aggregated polymer vesicles with an inner aqueous core (PEO-b-PTTAMA) have been reported for pH triggered exhibited great potential to incorporate oxygen-carrying release of enzyme AP.102 For stimuli-responsive macro- proteins. Following this work, several groups demonstrated molecule delivery, loading of polymersomes into layer-by- that polymersome could successfully encapsulate enzymes, layer polymer capsules has been reported.103 The polymeric including glucose-oxidase and oxygen transport proteins, like vesicles were formed from diblock copolymer poly(oligo- Hb and Mb, into polymersomes while retaining their − (ethylene glycol) methacrylate)-b-poly(2-(diisopropylamino)- bioactivity.41,42,85,133 135 Although these studies proved ethyl methacrylate) (POEGMA-b-PDPA) due to its amphi- successful loading of functional proteins into polymersome philic nature at physiological pH. The polymersomes cores, the encapsulation efficiencies were very low due to encapsulated with pDNA were further coated with a layer- hydrodynamic repulsion of the inward PEG chains against the by-layer polymer capsule. The release of pDNA was observed proteins.136,137 A PEG-g-PCL-based Hb-encapsulating poly- in response to pH changes from physiological to endocytic mersome was reported with a slightly improved encapsulation conditions. This study demonstrated a novel multicomponent efficiency of 26% compared with the thin-film hydration- nanocarrier with tailored degradation kinetics due to the extrusion method,41,134 Its potential application as an artificial combination of both polymersomes and polymer capsules. oxygen carrier for transfusion was reported as well.71 Photoresponsive polymeric vesicles were developed from The encapsulation of therapeutic protein insulin was photocleavable amphiphilic block copolymers as light-triggered investigated in PLA-b-pluronic-b-PLA (PLA-b-F127-b-PLA) nanocarriers.104 Under UV irradiation, these vesicles disinte- vesicles.140 Encapsulated insulin led to a hypoglycemic effect

K https://dx.doi.org/10.1021/acs.biomac.9b01754 Biomacromolecules XXXX, XXX, XXX−XXX Biomacromolecules pubs.acs.org/Biomac Review after oral administration to diabetic mice, demonstrating the taken up by three (HeLa, Jurkat, and HEK 293) cell types, and potential of these vesicles as protein carriers for in vivo delivery. their uptake pathway and cellular fate was investigated. The in Improved delivery was observed when insulin was loaded in vitro investigation of HRP loaded TAT-polymersomes neutral PEG-b-PBD polymersomes.20 Fluorescence microscopy demonstrated that 42% of the enzyme activity was still imaging of these vesicles revealed that encapsulated protein preserved after 16 h compared to free enzyme, with a half-life was retained in the aqueous lumen (Figure 3a). Also, insulin- of roughly 1 h. These studies together demonstrate progress loaded vesicles prepared by film rehydration were stable in toward therapeutic use of actively targeted polymersomes blood, maintaining the insulin secondary structure without decorated with ligands. significant loss (Figure 3b). Recently, galactose-decorated and GrB loaded chimeric polymersomes were designed and developed from galactose-b- PEG-b-PCL (Gal-b-PEG-b-PCL), asymmetric PEG-b-PCL-b- poly(2-(diethylamino)ethyl methacrylate) (PEG-b-PCL-b- PDEA), and PEG-SS-PCL for efficient intracellular protein delivery.141 CC and GrB (apoptotic protein)-loaded Gal- decorated reduction-sensitive chimeric polymersomes effectively caused apoptosis in HepG2 cells in vitro. Novel, degradable, and pH-sensitive chimeric polymersomes surface decorated with anisamide and termed Anis-CPs, were also investigated for targeted delivery of GrB to lung cancer cells.155 Anisamide end-capped PEG-b-poly(2,4,6- trimethoxybenzyli- dene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-b-poly- (acrylicacid) (Anis-PEG-b-PTTMA-b-PAA) and PEG-b- PTTMA-b-PAA copolymers were used to fabricate polymersomes. The in vitro investigation of GrB-loaded Anis-CPs in H460 cells demonstrated increased antitumor efficacy with increasing anisamide content (from 0 to 80%). The induced apoptosis was widespread, as demonstrated by flow cytometry. The high potency and receptor specificity in H460 and PC-3 cells of this system was particularly interesting for targeted therapy of lung cancers. Additionally, a cRGD and fusogenic GALA peptide-decorated, dually functionalized polymersome was developed for highly efficient delivery of protein into the cytosol.145 These nanocarriers enabled ανβ3-specific binding and enabled cytosolic delivery of apoptotic protein CC in A549 human lung cancer cells. These studies highlighted the functionalization potential of polymersomes for cytosolic Figure 3. Encapsulation of therapeutic proteins into polymersomes. delivery of proteins. (a) Fluorescent micrographs of fluorescent PKH26 (red) labeling the fl To improve the therapeutic potential of polymersomes, membrane of a giant OB2 polymersome and uorescein-labeled multi-stimuli-responsive carriers have also been developed. In bovine insulin (green). Scale bar = 10 μm. (b) CD results for recombinant human Zn-insulin after incubation for 8 h at 4 and 60 °C this regard, dual-bio responsive polymersomes, which were indicates that the film rehydration technique does not result in the highly sensitive to both intracellular pH and reductive loss of insulin secondary structure. Reprinted from ref 20. Copyright environment, were developed for efficient encapsulation and 2008, with permission from Elsevier. triggered release of proteins, such as fluorescein isothiocyanate (FITC)-labeled BSA (FITC-BSA) and CC (FITC-CC), into 3.1. Applications in Cancer Delivery. To broaden the cells cytosol.152 The polymersomes were prepared from PEG- scope of polymersomes for therapeutic protein delivery, several SS-poly(2- (diethyl amino)ethyl methacrylate) (PEG-SS- studies have demonstrated their anticancer potential in vitro. PDEA) diblock copolymers with PDEA, offering efficient To improve the delivery of protein-loaded polymersomes to encapsulation and stabilization due to its cationic nature and their target site, efforts have been made to prevent nonspecific endosomal buffering capacity. The in vitro investigation of CC distribution. PEO-b-PBD-based vesicles encapsulating tumor loaded PEG-SS-PDEA polymersomes in MCF-7 cells proved necrosis factor-α (TNF-α) and surface decorated with PR-b, markedly enhanced apoptosis compared to free protein and ff α β 138 an e ective 5 1 targeting peptide, were formulated. These reduction-insensitive controls (i.e., CC-loaded PEG-b-PDEA polymersomes were formed by aqueous film rehydration and polymersomes). In a similar study, reduction- and pH-sensitive the giant polymersomes created were subsequently extruded cross-linked polymersomes based on triblock polymer PEG-b- through a membrane to obtain particles less than 100 nm in PAA(SH)-b-PDEA were formed for efficient intracellular size, while retaining the protein encapsulant. PR-b decorated delivery of apoptotic proteins to cancer cells and noncancerous polymersomes encapsulating TNF-α demonstrated improved cells.154 The antitumor activity of polymersomes was studied cytotoxicity compared to free TNF-α and RGD peptide- in various cancer cell lines, such as MCF-7 and HeLa cells, decorated polymersomes in human prostate cancer cells which exhibited cell viability of 11.3 and 8.1%, respectively. In (LNCaP). A TAT-presenting semiporous membrane polymer- a noncancerous cell line, 293T, lower cytotoxicity (52.7%) was some were formed by PS-b-PIAT and 10 wt % of TAT attributed to slower uptake of polymersomes and lower covalently linked to a polystyrene-b-PEG-oxanorbornadiene glutathione concentration. In another study, reduction and (PS-b-PEG-TAT).158 Polymersomes loaded with GFP were temperature dual-bioresponsive cross-linked polymersomes

L https://dx.doi.org/10.1021/acs.biomac.9b01754 Biomacromolecules XXXX, XXX, XXX−XXX Biomacromolecules pubs.acs.org/Biomac Review

Figure 4. ANG-directed redox-responsive polymersomes (ANG-PS) for selective delivery of SAP to orthotopic human glioblastoma xenografts in mice. Reprinted with permission from John Wiley and Sons from ref 144. Copyright 2018 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim. were developed from two thermosensitive triblock copolymers, virus-mimicking polymersomes, surface-decorated with angio- PEG-b-PAA-b-PNIPAM and PEG-b-PAA-b-PNIPAM.159 The pep-2 (ANG-PS), have been reported for selective and efficient in vitro investigation of CC-loaded and cystamine-cross-linked delivery of potent natural protein toxin, saporin (SAP) to polymersomes in MCF-7 cells confirmed markedly enhanced human glioblastoma xenografts in mice144 (Figure 4). In vitro, apoptosis compared to free protein and the reduction SAP-loaded polymersomes displayed marked antitumor insensitive controls due to maintenance of biological activity activity in U-87 MG human glioblastoma cells (IC50 of 30.2 of macromolecules after release from nanostructures. nM). In vivo, they exhibited higher transcytosis across the For potential therapeutic applications, the nanocarriers need blood brain barrier (BBB) (for 20% angiopep density) and to be extensively tested in preclinical animal studies to enhanced tumor accumulation (1.71% ID/g) in mice bearing determine circulation times, maintenance of therapeutic U-87 MG orthotopic glioblastoma tumors, which resulted in activity, immunogenicity, and overall therapeutic effect. In effective inhibition of tumor growth and improved survival rate this regard, Zhong and colleagues have investigated the with little side effects. In a separate study, ApoE decorated potential of chimeric polymersomes in vivo. They designed chimeric polymersomes (ApoE-CP) loaded with SAP dis- pH-responsive chimeric polymersomes decorated with 2-[3-[5- played highly specific and potent antitumor affects both in vitro amino-1-carboxypentyl]-ureido]-pentanedioic acid (Acupa), in U-87 MG cells and in vivo in orthotopic U-87 MG called Acupa-CPs, for efficient delivery of therapeutic proteins glioblastoma.143 Compared to ANG counterparts, ApoE CPs CC and GrB into prostate cancer cells (LNCaP).146 increased BBB transcytosis 2.2-fold as well as enhanced GBM Preliminary in vivo studies of these PSMA-targeting pH- targeting. sensitive degradable chimeric polymersomes (Acupa-CPs) in To further demonstrate the therapeutic potential of nude mice revealed prolonged circulation time with an polymersomes, a cell-selective penetrating and reduction- elimination half-life of 3.3 h. The first report of polymersomes responsive polymersome (CPRPs) was developed for efficient for cancer-targeted protein therapy in vivo utilized anisamide- and targeted delivery of GrB in vivo to orthotopic human lung 76 functionalized bioresponsive chimaeric nanopolymersomes tumors. In vitro, these CPRPs displayed high potency (IC50 formulated from triblock copolymers PEG-b-poly(N-2- hydrox- of 20.7 nM) in lung cancer cells (A549). In vivo, ypropyl methacrylamide-g-lipoic acid)-b-poly(acrylic acid) functionalization with cell-selective penetrating peptide (PEG-b-P(HPMA-LA)-b-PAA) and Anis-PEG-b-P- enhanced tumor accumulation compared with nonfunctional- (HPMALA)-b-PAA.156 These nanoparticles had prolonged ized counterparts without influencing blood circulation time circulation time with an elimination half-life of 6.3 h and (6.6 h) and demonstrated complete tumor growth inhibition remarkable tumor accumulation (11.5% ID/g). Therapeutic within 20 days. Recently, asymmetric PEG-b-poly(α-amino- doses (6.24 nmol of GrB/kg) of these polymersomes, given palmitic acid)-b-poly(L-aspartic acid) triblock copolypeptide either as a single injection or four divided injections to mice, (PEG-b-PAPA-b-PAsp) was surface-functionalized with RGD effectively inhibited H460 tumor growth and improved survival to form targeted chimaeric vesicles called “lipopepsomes” rate compared to untreated group. Recently, redox-responsive (cRGD-CLP) for in vivo delivery of SAP.162 In vitro, these

M https://dx.doi.org/10.1021/acs.biomac.9b01754 Biomacromolecules XXXX, XXX, XXX−XXX Biomacromolecules pubs.acs.org/Biomac Review vesicles enhanced internalization in A549 lung cancer cells demonstrated effectiveness as a prophylactic cancer nano- α β overexpressing v 3 by twofold compared with nonfunction- vaccine by delaying tumor occurrence and prolonging survival alized controls. In vivo, they showed remarkable accumulation through delivery of antigen and adjuvant in a spatiotemporal in cancerous lungs of mice (7.73% of ID/g) and higher manner to induce a synergistic antitumor immune response. potency in treating A549 lung tumors with improved survival 3.3. Other Therapeutic Applications. A number of rates. studies have recently reported the delivery of protein A reduction-responsive chimaeric polymersome (RCP) macromolecules to the CNS. A pH-sensitive polymersome encapsulating GrB and surface decorated with hyaluronic constructed from diblock polymer poly[oligo(ethylene glycol) acid (HA) (HA-RCP-GrB) has been reported recently for methyl methacrylate]-b-poly(2-(diisopropylamino)ethyl meth- targeted protein therapy of multiple myeloma.147 In vivo acrylate) (POEGMA-b-PDPA) and surface functionalized with biodistribution of these polymersomes demonstrated enhanced Angiopep-2 (A-EP) was reported for CNS delivery of large accumulation in bone marrow of multiple myeloma orthotopic macromolecule IgG to both CNS parenchyma and CNS model compared with nontargeted polymersomes demonstrat- cells.164 In vitro, these vesicles showed transcellular movement ing their selective delivery. Therapeutic evaluation revealed across a tight endothelial layer and in a 3D model of the BBB significant survival benefit and effective treatment of multiple via an active transport mechanism. In vivo, these vesicles myeloma. protected IgG from systemic degradation, penetrated the 3.2. Applications in Vaccine Delivery. The aqueous parenchyma, and delivered IgG to neurons and glial cells. A interior of polymersomes permits safe transport of antigens diblock copolymer, PEG and Ketal-modified polyserine (PEG- and adjuvants without necessitating their chemical modifica- poly-Ser-Ketal)-based polymersomes were reported for release tion. Designing new adjuvant formulations and antigen of insulin in a glucose responsive manner.160 A single delivery systems could assist in matching the characteristic subcutaneous injection of these vesicles facilitated stabilization needs of target recipients. Polymersomes offer potential of blood glucose levels in the normoglycemic state (<200 mg/ benefits by encapsulating unmodified protein antigens along dL) for up to 5 days. with small molecule adjuvants due to their higher stability and Our group has demonstrated delivery of β-galactosidase (β- tunable properties. Initial work has involved the association of gal) by apolipoprotein E (Apo E)-decorated PEG-b-PLA antigen with polymersomes as hybrid assemblies. The polymersomes as enzyme-based therapy to the brain in polypeptide-b-peptide copolymer-based vesicles loaded with lysosomal storage disease.58 These pH-responsive polymer- influenza hemagglutinin (HA) showed enhanced immunoge- somes showed burst release at acidic pH (4.8) of the lysosome nicity of the antigen in vivo, with polymersomes acting as an and restored β-gal activity in GM1 gangliosidosis cells to adjuvant.163 Stimuli-responsive oxidation-sensitive polymer- normal levels. This system is being tested in vivo in a feline somes based on PEG-b-PPS were developed for endocytic model of GM1 gangliosidosis. Recently, triblock copolymer- delivery of antigenic protein or TLR agonist adjuvants into based vesicles formed by PMOXA-b-PDMS-b-PMOXA, were dendritic cells to induce antigen-specific cell-mediated immune reported for efficient loading and delivery of neurotrophic responses.148 Encapsulation and release of model antigen proteins.139 The study confirmed coencapsulation of unstable ovalbumin from these vesicles resulted in activation of splenic neurotrophins, a large protein, CD109 (180 kDa), and small dendritic cells (DCs) and priming of OT-I CD8+ T cells in molecule fluorescent drug curcumin. Furthermore, neuronal vitro due to antigen cross-presentation through MHC I. The in targeting was enabled by covalently attaching N-Methyl-D- vivo potential of this platform was investigated to understand aspartate receptor (NMDAR) agonists and antagonists. In the nature of the immune response.149 Mice immunized with vitro, the vesicles were nontoxic to murine macrophages, these polymersomes had several-fold higher frequencies of human fibroblasts, and 3D models of the peripheral nervous antigen-specific CD4+ T cells in the lungs, spleen, and lymph system (DRG explants), meriting their further investigation in nodes compared to free antigens due to efficient delivery to the in vivo animal models. MHC II presentation pathway. On the contrary, polymersomes The controlled release of angiogenic factors also has with surface-conjugated antigen were more efficiently delivered potential applications in tissue regeneration. For such progress, to the MHC I presentation pathway and induced CD8+ T cell amphiphilic graft copolymer-based polymeric vesicles were immunity. This difference was attributed to variations in reported for the delivery of bioactive vascular endothelial 161 biodistribution and targeting of different subcellular compart- growth factor (VEGF). Poly(L-lysine)−g-poly(lysine- ments and/or antigen-presenting cells (APCs). A polymer- (arachidic acid)) copolymer was used for the effective some-based adjuvant and antigen delivery system loaded with condensation and encapsulation of VEGF without influencing CL075 (selective TLR8 agonist) and Mycobacterium tuber- size and surface charge of polymersomes. Compared to culosis antigen Ag85Bp25, termed CL075:Ag85Bp25-PS, nongrafted vesicles, arachidic acid-grafted polymersomes induced an antigen-specific CD4+ adaptive immune response increased the release of VEGF (2.23 ng/mL) and transfection in vivo in humanized TLR8 transgenic neonatal mice efficiency with little toxicity due to peptide-based nature of (huTLR8Tg).150 This study demonstrates the potential of vesicles. Therapeutic outcomes are expected to improve with polymersomes as an alternative to Bacillus Calmette-Gueriń the design and development of theranostics systems. (BCG) vaccines in the development of new early life vaccines. Theranostic vesicles have been developed for magnetic Recently, a well-designed vesicle-based nanovaccine was resonance imaging and anticancer drug delivery.142 In one developed by coencapsulating antigen and adjuvant in a single study, amphiphilic diblock copolymer PEG-b-PLGA was used vesicle and delivering them to the same dendritic cells to encapsulate BSA-gadolinium (BSA-Gd) complexes. Imaging (DCs).72 These cationic lipid hybrid polymersomes which experiments both in vitro and in vivo revealed excellent T1- were surface decorated with mannose receptors, efficiently weighted magnetic resonance images. The simultaneous adsorbed OVA antigen on the surface via electrostatic delivery of anticancer drug doxorubicin along with BSA-Gd interaction and also inside the lumen of vesicles. This platform resulted in slower release of doxorubicin compared to vesicles

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Table 4. Polymersomes-Based Vesicles for Delivery of Nucleic Acid-Based Macromolecules

polymer fabrication method cargo size remarks ref A. polymersomes for the delivery of DNA PEG-b-PLL/Chol probe sonication pCMV β-Gal 375−593 nm amphiphilic amino acid-based polymer 166 PEG-b-PO/Chol vesicles PEG-b-PLL/Chol/DSPE-PEG-GA probe sonication pCMVluc, 330−800 nm reduced toxicity and improved 167 PEG-b-PO/Chol/DSPE-PEG-GA pEGFP-C1 applicability in vivo PEG-b-PEI-b-PA/Chol probe sonication pCMVbeta, improved biocompatibility 168 pEGFP-C1 PBD-b-P4VPQ three-step single emulsion pUC18 2−15 μmefficient encapsulation of charged payloads 38 PBD-b-P4VPQ three-step single emulsion pUC18, pEGFP- 2−15 μmefficient encapsulation of charged payloads 169 N1 PMPC-b-PDPA film rehydration pEGFP 200−400 nm pH induced encapsulation of DNA 170 PMPC-b-PDPA film rehydration pEGFP, pGL3 200−400 nm pH induced encapsulation of DNA 171 Chol-PEI-b-OA (LPSA) direct dissolution of pEGFP 110 nm one-step assembly and cytosolic delivery 172 copolymer PEG-b-DPA-phz (PEDP) solvent-free, solvent- pmIL_12, pVec 206 nm systemic injection of vesicles for plasmid 173 displacement delivery folate−PEG-b-ImHeMA-b-(GMA) direct dissolution of dsDNA 10 mer 139 nm pH responsive vesicles for nucleic acids 174 copolymer delivery B. polymersomes for the delivery of siRNA PEG-b-PCL, PEG-b-PLA, and PEG-b- solvent injection siRNA, AON 100 nm nonionic vesicles for gene delivery 175, PBD applications 176 fi PB-b-PEO and PB-b-PEO-N3 lm rehydration siOrai3 249 nm model system for targeted gene delivery 177 PEG-b-PLA film rehydration Bcl-xL siRNA 169 nm codelivery of genes and drugs via single 178 vesicle PEO-b-PDPA-b-PAA direct dissolution of siFITC, miRNA- 232 nm pH-sensitive vesicles for gene and drug 179 copolymer 429 siRNA codelivery PAsp(DIP)-b-PLys direct dissolution of siFITC 203 nm pH-sensitive peptides-based vesicles for 180 copolymer drug and gene codelivery PEG-b-PCL-b-DEX phase-guided assembly siFAM, 145 nm nanocarrier for efficient gene delivery in 181 siTAMRA, vivo siGL3 PEG-b-pImHeMA-b-pGMA direct dissolution of ds-DNA, ds- 273 nm pH controlled encapsulation of genetic 182 copolymer siRNA macromolecules PEG-b-P(TMC-co-DTC)-b-PEI, cNGQ- solvent exchange method siPLK1 175 nm chimeric vesicles for targeted cancer 183 PEG-b-P(TMC-co-DTC) siRNA therapy PEG-b-P(TMC-co-DTC)-b-PEI, ANG- solvent exchange method siPLK1 115 nm chimeric vesicles for targeted cancer 184 PEG-b-P(TMC-co-DTC) siRNA therapy PEO-b-P(NIPAM-stat-CMA-stat-DEA) direct dissolution followed FAM-siRNA, 462 nm dual gating gene vesicles for on-demand 185 by UV curing GFP-pDNA delivery siRNA-SS-PNIPAM heating Pgp-siRNA 135 nm cation free nanostructures for controlled 186 loading and gene delivery PEG-b-PAPA-b-PLL, CPP33-PWG-b- solvent exchange method siPLK1 90 nm chimeric vesicles for selective delivery of 187 PAPA siRNA without BSA complexes possibly due to electrostatic and modification of poly-L-lysine and poly-L-ornithine with hydrophobic interactions between the drug and the protein.165 palmitoyl and methoxy PEG (mPEG) were efficient delivery vehicles for gene transfer in vivo (to lungs and liver).166 4. POLYMERSOMES FOR NUCLEIC ACID DELIVERY Increased expression of luciferase by delivering pCMVluc in Transport of nucleic acid-based materials into target cells has HepG2 cells was observed when a galactosamine targeting been regarded as a novel method of disease therapy. In this ligand was bound to PEO chains.167 A similar modification regard, polymersomes have gained increasing interest due to with polyethylenimine (PEI) created amphiphilic polymer their void, hydrophilic inner core for efficient encapsulation, PEG-b-PEI-b-PA, which formed vesicles and mediated GFP and their hydrophobic membrane for improved protection and transgene expression in the liver after intravenous injection.168 controlled release, depending on the nature of the constituent Cationic diblock copolymer poly(butadiene-b-N-methyl-4- polymers. They also provide a means of cell entry and vinylpyridinium) PBD-b-P4VPQ-based vesicles were formu- intracellular release of these sensitive payloads. Various nucleic lated for the encapsulation and controlled release of macro- acids macromolecules (pDNA, AON, siRNA) that have been molecules, such as double-stranded DNA fragments.38 These developed with polymersomes in recent years for both in vitro cationic vesicles enabled encapsulation of model plasmids, and in vivo delivery are listed in Table 4. ff 4.1. DNA Delivery. Initial designs of polymeric vesicles for release of payloads by two di erent mechanisms (either by DNA delivery were inspired by DNA-polycation polyplexes,188 osmotic rupture or by electrostatic screening), and demon- focused on modifications of charged poly amino acids and PEI- strated reverse transfection of plasmids into HeLa cancer cells, 169 based polymers to make them less toxic for gene delivery with cells growing on top of the expression vector. A applications. One study showed that poly(amino acid) polysaccharide-block-polypeptide copolymer, i.e. dextran-b- (poly(AA))-based polymer vesicles formed by covalent poly(γ-benzyl L-glutamate) (DEX-b-PBLG) block copolymer,

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Figure 5. Encapsulation of AONs and siRNAs in polymersomes. (a) Fluorescence images of FITC-AON (green)-loaded fluorescently labeled polymersomes (red). (b) Fluorescence images of FITC-siRNA (green)-loaded into polymersomes (red). Reprinted from ref 175. Copyright 2008, with permission from Elsevier. was reported to mimic viral capsids, with potential for gene N,N-diisopropylethylenediamine (DPA) hydrophobic groups delivery.189 and mPEG tails were formulated for systemic delivery of A pH-sensitive diblock copolymer-based vesicle was formed murine plasmid pmIL-12.173 The encapsulation of plasmid from PMPC-b-PDPA enabled physical encapsulation of pDNA DNA was achieved by both physical and electrostatic into vesicular structures by simply adjusting the pH due to interactions, and polymersomes demonstrated serum stability tertiary amine groups of PDPA chains which are hydrophobic and hemocompatibility. Intravenous injection of these vesicles at physiological pH but become hydrophilic in mild acidic achieved significant tumor suppression in mice bearing CT-26 conditions.170 The pDNA encapsulation efficiency was 20% colon carcinoma, compared to vector plasmid (pVec) vesicles with vesicle sizes of 200−400 nm. Upon disassembly of and saline control. The structural similarity of DPA with nanostructures in weakly acidic conditions, strong interactions DMAEA offered these polyphosphazene-based polymersomes between the PMPC-b-PDPA diblock copolymer and the DNA potential biodegradability. In another study, a family of pH- led to the formation of complexes. These polymersomes responsive triblock copolymers were evaluated for delivery of exhibited excellent colloidal stability at room temperature for nucleic acids.174 The polymeric vesicles were able to up to three months. In vitro, in Chinese hamster ovary (CHO) encapsulate short 19-mer dsDNA, demonstrated pH-respon- and human dermal fibroblast (HDF) cells, polymersomes sive release, and were well tolerated by cells. Folate- mediated lower cytotoxicity and higher levels of GFP functionalized vesicles exhibited increased uptake in KB cells expression in the cytosol compared with lipofectamine and (overexpress folate receptors) compared to MCF-7 cells. calcium phosphate particles. The platform protected pDNA 4.2. siRNA Delivery. Discher and colleagues demonstrated and enabled sufficient delivery due to physical encapsulation at the delivery of siRNA and antisense oligonucleotides by neutral pH and stealth properties provided by the PMPC chain biodegradable, and thus more relevant, block copolymers on the exterior of the vesicles. These polymersomes were also including PEG-b-PCL, PEG-b-PLA, and PEG-b-PBD-based able to maintain the activity of encapsulated DNA for at least 2 nonionic polymersomes.175 The encapsulation efficiencies weeks without significant loss of pDNA, and successfully were found to be 30% for siRNA in PEG-b-PLA polymersomes delivered luciferase encoding plasmid (pGL3) into HDF and 20% for AONs in PEG-b-PCL polymersomes (Figure 5). cells.171 The vesicles (100 nm) were believed to transform into micelles In an effort to improve the cytosolic delivery of polymeric (60 nm) to release their oligos.9 The vesicles encapsulating vesicles, a triblock graft copolymer, lipopolysaccharide-amine siRNAs targeting lamin A/C exhibited knockdown in A549 (LPSA), with both amphotericity and amphiphilicity, was lung cancer epithelial cells in vitro, while AON-loaded vesicles developed.172 The polymer block had two oppositely charged were taken up by myotubes, and released AON with nuclear hydrophilic blocks, cationic PEI and anionic oxidized alginate localization, leading to dystrophin expression in mdx mice. In (OA), and a hydrophobic block grafted on PEI which was another study, AONs were delivered by biodegradable polymer constituted by cholesteryl. The cytosolic localization of pDNA vesicles formulated from PEG-b-PBD.176 These polymersomes was observed in mesenchymal stem cells after delivery via allowed encapsulation, uptake, in vitro release, and delivery of polymersomes with high transfection efficiency; more than AONs into C2C12 myotubes. Inside the cells, polymersomes 95% of cells expressed GFP (intact genes were delivered to were found close to the nucleus, and AONs were released and cytosol first followed by nuclear entry). Amphiphilic delivered to the nucleus. In mdx-dystrophic mice, intra- polyphosphazene-based polymersomes with weakly cationic muscular (IM) injection of these polymersomes into the hind

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Figure 6. Targeted delivery of siRNAs by cNGQ peptide-directed, reversibly cross-linked chimeric polymersomes (cNGQ/RCCPs) to orthotopic lung tumors in mice. Reprinted with permission from John Wiley and Sons from ref 183. Copyright 2017 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim. limbs led to efficient nuclear uptake and dystrophin expression. demonstrating their potential application for liver disease The absence of cationic groups in these vesicles suggested therapy. broader application of these nanocarriers. Simultaneous delivery of more than one therapeutic has Self-assembled polymeric vesicles, termed oligomersomes been shown to be an effective strategy to overcome deficiencies (TCOsomes), were formulated from amphiphilic α-tocopherol of treatment based on single therapeutic delivery platforms. oligochitosan conjugates for siRNA delivery.190 The siRNA Diblock copolymer (mPEG-b-PLA)-based polymersomes complexation on the TCOsomes surface caused thickening of (CPSomes) were designed and investigated for codelivery of 178 the membrane. In vitro, these nanocarriers significantly Bcl-xL siRNA and doxorubicin (DOX). The siRNA was enhanced cellular uptake of siRNA in human epidermal encapsulated inside the core of these vesicles with an ffi carcinoma cell line KB cells and reduced target protein encapsulation e ciency of 55%, while doxorubicin was expression compared to free siRNA. In a mouse KB tumor embedded in the membrane. The in vitro transfection of xenograft model, siMcl-1-encapsulated polymersomes substan- MKN-45 and MKN-28 human gastric cancer cell lines with tially reduced expression of Mcl-1, demonstrating effective combination therapy-loaded vesicles revealed increased levels of Bax expression while expression of Bcl-xL was decreased, silencing and preventing tumor growth. ffi Polymer vesicles consisting of peptide-functionalized PBD-b- demonstrating the synergistic e cacy (by inducing additive PEO were reported for efficient encapsulation and delivery of cell killing or by increasing chemotherapeutic sensitivity) of the 177 codelivery strategy. pH-sensitive block copolymer vesicles siRNA. The siRNA was physically encapsulated into surface-decorated with monoclonal antibody anti-EpCAM aqueous lumen with an efficiency of 51%. These peptide (epithelial cell adhesion molecule), were developed for (PR-b) functionalized vesicles encapsulating siOrai3 mediated targeting cancer stem cells (CSCs).179 Polymersomes based moderate Orai3 knockdown in T47D and MCF710A cells in on the triblock copolymer PEO-b-poly[2-(diisopropylamino)- vitro. This was attributed to release of payloads in early ethyl methacrylate]-block-poly(acrylic acid) (PEO-b-PDPA-b- endosomes and their escape from acidic organelles. Although PAA) were able to codeliver anticancer drug (doxorubicin)and the OB block was nonbiodegradable in nature, this study paved siRNA (FITC-siRNA) to overcome self-renewal and tumor the way for future improvements and advancements for siRNA producing capabilities of CSCs by decreasing expression of cell delivery by polymeric vesicles. Triblock copolymer-based promoting oncogene miR-429. pH-responsive diblock copoly- vesicles formed by PEG-b-PCL-b-DEX (PPD) and protamine 181 peptide-based vesicles were also reported for codelivery of were developed for delivery of nucleic acids. siRNAs were chemotherapeutic drugs and siRNA.180 The well-defined condensed with protamine (cationic polypeptide to mediate diblock copolypeptides (PAsp(DIP)n-b-PLysm) self-assembled condensation of nucleic acids) and encapsulated with high into vesicles in an aqueous environment and oligonucleotides efficiency into polymersome cores due to the favored partition interacted with the exterior of these vesicles for codelivery and of protamine into the dextran cavity rather than the PEG shell. rapid intracellular release of DOX and siRNA into HepG2 In SMMC-7721 cells, these vesicles enabled high cellular cells. To allow better control over tissue and cell localization, uptake, intracellular delivery of siRNAs, and efficient (40%) pH-responsive polymersomes were generated from a triblock silencing of gene expression. In vivo biodistribution of these copolymer PEG-b-polyimidazole-hexyl methacrylate-b-polygly- carriers suggested preferential accumulation in the liver, cerolmethacrylate (PEG-b-pImHeMA-b-pGMA) for pH-con-

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Figure 7. Macromolecular shuttle designed from dually gated polymersomes as a gene delivery carrier. Reproduced with permission from ref 185. Copyright 2018 American Chemical Society. trolled association with DNA and encapsulation of functional penetrating peptides (CPP33) which led to selective delivery siRNA.182 The oligonucleotides were encapsulated (5.4 mol of siRNAs into A549 human lung tumors in vivo.187 siRNA was %) into polymersomes and due to the presence of imidazole- efficiently encapsulated into the watery core of these vesicles containing side chains, endosomal escape occurred. Folate by electrostatic complexation with PLL. targeted polymersomes mediated 31 and 23% knockdown of In an effort to efficiently load and control the release of − luciferase and Hsp90 protein expression, respectively, in B16 sensitive nucleic acids, a polymersome with a “boarding gate” F10 cells. “ ” 185 ffi and a debarkation gate in its membrane was developed, as Adapted for their in vivo therapeutic e cacy for protein shown in Figure 7. A block copolymer composed of PEO-b- delivery, virus-mimicking chimeric polymersomes were re- poly[N-isopropylacrylamide-stat-7-(2-methacryloyloxyethoxy)- cently reported for siRNA delivery in cancer therapy,183 as 4-methyl coumarin-stat-2 (diethylamino) ethyl methacrylate], shown in Figure 6. The vesicles were assembled from either an asymmetric biodegradable triblock copolymer, PEG-b-poly- PEO-b-P(NIPAM-stat-CMA-stat-DEA), was used for vesicle (trimethylene carbonate-co-dithiolane trimethylene carbonate)- synthesis. The dually gated heterogeneous membrane was b-PEI (PEG-b-P(TMC-co-DTC)-PEI), or a cNGQ peptide- formed from a pH-sensitive P(NIPAM-stat-CMA-stat-DEA) functionalized diblock copolymer, cNGQ-PEG-b-P(TMC-co- block, which opened at room temperature, the temperature- DTC). siRNA was successfully loaded into the lumen of these controlled “boarding gate,” to encapsulate siRNA and plasmid vesicles. In vitro, polymersomes mediated efficient uptake of DNA, and released payloads inside the cell by the proton siRNA into α3β1-integrin-overexpressing A549 lung cancer sponge effect, the “debarkation gate.” This vesicular platform cells and induced potent and sequence-specific gene silencing exhibited efficient gene transfection both in vitro and in vivo, due to rapid release of siRNA. In a mouse model of orthotopic suggesting that dually gated polymersomes are an excellent A549 human lung cancer, siPLK1-loaded polymersomes strategy for controlled shuttling of genetic biomacromolecules. allowed prolonged circulation, enhanced tumor accumulation, An siRNA-based vesicle called a “siRNAsome” was reported andsuppressedtumorgrowth with improved survival as a cation-free nanostructure which consisted of a hydrophilic compared with nonfunctionalized vesicles and saline control. siRNA shell, a hydrophobic median layer sensitive to thermal In another study, these chimeric polymersomes were surface and intracellular reduction, and an aqueous empty interior functionalized with angiopep-2 peptide (ANG-CP) to boost core.186 The nanostructures were constructed from siRNA-SS- RNAi therapy in brain tumors.184 In vivo evaluation PNIPAM, and inherent thermal and reduction responsiveness demonstrated that this vesicular platform induced strong antiglioblastoma effects, with a significantly improved survival enabled loading and release of payloads in a controlled time compared with nonfunctionalized vesicles and saline manner. Coencapsulating these polymersomes with siRNAs control in glioblastoma tumor-bearing mice. Recently, vesicle targeting P-glycoprotein (Pgp) and doxorubicin enabled nanostructures named “chimeric lipopepsomes” were devel- synergistic therapeutic activity in multidrug resistant (MDR) oped from PEG-b-poly(α-amino palmitic acid)-b-poly(L- MCF-7 cancer cells and in MCF-7 tumor-bearing mice. These lysine) (PEG-b-PAPA-b-PLL), an asymmetric triblock copoly- siRNAsomes can function as a versatile nanoplatform for mer, where the PAPA segment allowed strong lipid−lipid delivering immobilized siRNAs without transfection agents, packing. These vesicles were functionalized with cell- other drugs, or macromolecules.

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5. CONCLUSIONS AND FUTURE PERSPECTIVES ment, polymersome-based protein and nucleic acid delivery for An ideal nanocarrier for efficient delivery of macromolecules to therapeutics will be a reality in the near future. cells is envisioned to fulfill the following criteria: (a) deliver the macromolecules in active conformation; (b) offer sufficient ■ AUTHOR INFORMATION protection from harsh environments encountered in vivo, such Corresponding Authors as but not limited to degradation by proteases and nucleases Angela Alexander-Bryant − Department of Bioengineering, during circulation; (c) demonstrate limited toxicity to cells; Clemson University, Clemson, South Carolina 29634, United (d) accumulate and deliver the therapeutic at the target site; States; Email: [email protected] ffi and (e) enable e cient escape from endo/lysosomal compart- Jessica Larsen − Department of Chemical and Biomolecular ments within cells. The past decade has witnessed extensive Engineering and Department of Bioengineering, Clemson investigations and significant developments in engineering University, Clemson, South Carolina 29634, United States; polymeric vesicles for intracellular delivery of protein and orcid.org/0000-0003-2756-9523; Email: larsenj@ nucleic acid macromolecules. clemson.edu In contrast to conventional methods of macromolecule delivery, polymersomes offer highly specific and tunable Authors biologic responses with notably low toxicity, thereby rendering Shoaib Iqbal − Department of Chemical and Biomolecular them a safe and effective delivery alternative. In this Review, Engineering and Department of Bioengineering, Clemson the dynamics of assembly and design optimization for University, Clemson, South Carolina 29634, United States; macromolecule delivery was discussed. The influence of orcid.org/0000-0002-8778-4666 polymeric materials and preparation methods on polymersome Mark Blenner − Department of Chemical and Biomolecular formulation and properties for macromolecule delivery was Engineering, Clemson University, Clemson, South Carolina summarized, with the goal of establishing design criteria for the 29634, United States development of future nanocarriers. Current challenges faced Complete contact information is available at: by the macromolecule delivery nanocarriers include their poor https://pubs.acs.org/10.1021/acs.biomac.9b01754 cell penetration and inefficient intracellular transportation. The numerous design features of polymersomes, such as the overall Notes composition of polymer blocks, polymer topology, ability to be The authors declare no competing financial interest. formed in spherical or nonspherical vesicles and membrane thickness have contributed to their versatility and tunability, ■ REFERENCES making them an attractive platform for addressing many of the engineering and biological problems associated with the (1) Mitragotri, S.; Burke, P. A.; Langer, R. Overcoming the delivery of macromolecules. Finally, recent advances in challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat. Rev. Drug Discovery 2014, 13, 655. polymersome delivery of proteins and nucleic acids for various (2) Cheng, L.; Yang, L.; Meng, F.; Zhong, Z. Protein Nano- therapeutic applications were reviewed (Tables 3 and 4). therapeutics as an Emerging Modality for Cancer Therapy. Adv. 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