Morphology Regulation in Redox Destructible Amphiphilic Block Copolymers and Impact on Intracellular Drug Delivery

In two ABA type amphiphilic block copolymers (P1, P2), the hydrophobic B block consists of a bioreducible segmented poly(disulfide) (PDS), while poly‐N‐isopropylacrylamide (PNIPAM) or poly(triethyleneglycol)methylether‐methacrylate (PTEGMA) serve as the hydrophilic A blocks in P1 and P2, respectively, leading to the formation of polymersome and micelle, owing to the difference in the packing parameters. Both exhibit comparable doxorubicin (Dox) encapsulation efficiency, but glutathione (GSH) triggered release appears much faster from the polymersome than micelle owing to the complete degradation of the PDS segment in polymersome morphology unlike in micelle. Dox‐loaded polymers (P1‐Dox and P2‐Dox) exhibit minimum toxicity to normal cells like C2C12. By contrast, P1‐Dox shows excellent killing efficiency to the HeLa cells (cancer cell) (in which the GSH concentration is significantly higher). However, P2‐Dox reveals a rather poor activity even to HeLa cells. Fluorescence microscopy studies show comparable cellular uptake of P1‐Dox and P2‐Dox. But the polymersome entrapped dye escapes fast from the cargo and reach the nucleus, while the drug‐loaded micelle remains trapped in the perinuclear zone explaining the significant difference in the drug delivery performance of polymersome and micelle.     

How molecular internal‐geometric parameters affect PB‐PEO polymersome size in aqueous solution

Amphiphilic polybutadiene polyethylene oxide (PB‐PEO) is one of the best known chemistries to form stable vesicular morphologies, stated as polymersomes, in aqueous environment. Mimicking cell membranes, these structures self‐assemble in an “amphiphilic window” determined by 0.15 < f < 0.35 where f is the ratio between the hydrophilic block volume and the entire diblock volume. However the polymersome size distribution also depends on molecular weight (M_n) and in order to gain insight on how f and M_n together determine polymersome size, we prepared PB‐PEO diblock copolymers with different block lengths and analyzed vesicle morphology via Dynamic light scattering (DLS) and Freeze‐fracture transmission electron microscopy (FF‐TEM). We found three main regimes: high f / low M_n with polymersomes of mixed diameter, high f / high M_n with mainly large polymersomes and low f, with mainly small polymersomes. In the first region, the polymersomes are highly polydisperse. There is a tendency towards increased diameter with increasing f and M_n. Taken together our findings can help to identify how polymersome self‐assembly can be controlled to achieve size distribution specificity alleviating the need for subsequent tuning of size via extrusion. This can pave the way for cost‐effective upscaling of polymersome production for biomedical and biomimetic applications. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016 , 54, 699–708     

Formation and characterization of polymersomes made by a solvent injection method

In this article a solvent injection method is described for vesicle formation using poly(butadiene)‐ b‐poly(acrylic acid) diblock copolymers as the amphiphilic molecules. Vesicles composed of polymer bilayers are commonly referred to as polymersomes. Solvent injection is shown to be a rapid method for polymersome formation suitable to make large volumes of polymersome solution. The method can be manipulated to obtain a wide range of vesicle sizes depending on the polymer concentration and preparation conditions. Polymersome solutions in this study are characterized using dynamic light scattering (DLS), fluorescent microscopy, and electron microscopy. Polymersome sizes range from tens of nanometers to several microns. The membrane thickness of smaller polymersomes is found to lie between 14–20 nm. Larger polymersomes are found to have somewhat thicker membranes. The procedure involves the addition of polymers dissolved in an organic solvent to a stirred aqueous solution. The formation of polymersomes by this method is proposed to be governed by the limited mutual solubility of the two solvents and the simultaneous diffusion of solvent and water out of and in to initially formed organic solvent droplets. Copyright © 2007 John Wiley & Sons, Ltd.     

Dynamic Docking and Undocking Processes Addressing Selectively the Outside and Inside of Polymersomes

Increasing complexity and diversity of polymersomes and their compartments is a key issue for mimicking cellular functions and protocells. Thus, new challenges arise in terms of achieving tunable membrane permeability and combining it with control over the membrane diffusion process, and thus enabling a localized and dynamic control of functionality and docking possibilities within or on the surface of polymeric compartments. This study reports the concept of polymersomes with pH‐tunable membrane permeability for controlling sequential docking and undocking processes of small molecules and nanometer‐sized protein mimics selectively on the inside and outside of the polymersome membrane as a further step toward the design of intelligent multifunctional compartments for use in synthetic biology and as protocells. Host–guest interactions between adamantane and β‐cyclodextrin as well as noncovalent interactions between poly(ethylene glycol) tails and β‐cyclodextrin are used to achieve selective and dynamic functionalization of the inner and outer spheres of the polymersome membrane.     

Biocompatible functionalization of polymersome surfaces: a new approach to surface immobilization and cell targeting using polymersomes

Vesicles assembled from amphiphilic block copolymers represent promising nanomaterials for applications that include drug delivery and surface functionalization. One essential requirement to guide such polymersomes to a desired site in vivo is conjugation of active, targeting ligands to the surface of preformed self-assemblies. Such conjugation chemistry must fulfill criteria of efficiency and selectivity, stability of the resulting bond, and biocompatibility. We have here developed a new system that achieves these criteria by simple conjugation of 4-formylbenzoate (4FB) functionalized polymersomes with 6-hydrazinonicotinate acetone hydrazone (HyNic) functionalized antibodies in aqueous buffer. The number of available amino groups on the surface of polymersomes composed of poly(dimethylsiloxane)-block-poly(2-methyloxazoline) diblock copolymers was investigated by reacting hydrophilic succinimidyl-activated fluorescent dye with polymersomes and evaluating the resulting emission intensity. To prove attachment of biomolecules to polymersomes, HyNic functionalized enhanced yellow fluorescent protein (eYFP) was attached to 4FB functionalized polymersomes, resulting in an average number of 5 eYFP molecules per polymersome. Two different polymersome-antibody conjugates were produced using either antibiotin IgG or trastuzumab. They showed specific targeting toward biotin-patterned surfaces and breast cancer cells. Overall, the polymersome-ligand platform appears promising for therapeutic and diagnostic use.     

How preparation and modification parameters affect PB‐PEO polymersome properties in aqueous solution

The effect of formation and modification methods on the physical properties of polymersomes is critical for their use in applications relying on their ability to mimic functional properties of biological membranes. In this study, we compared two formation methods for polymersomes made from polybutadiene‐polyethylene oxide diblock copolymers: detergent‐mediated film rehydration (DFR) and solvent evaporation (SE). DFR‐prepared polymersomes showed a three times higher permeability compared to SE‐prepared polymersomes as revealed by stopped‐flow light scattering. SE‐prepared polymersomes broke down faster to structures <50 nm diameter when processed with extrusion, which was more pronounced at 5 mg mL^?1, compared to 10, 20, and 25 mg mL^?1. Our results indicate that the bilayer of SE‐prepared polymersomes has a lower apparent fluidity. We also investigated the role of n‐octyl‐β‐d ‐glucopyranoside (OG), a detergent typically used for reconstitution of membrane proteins into lipid bilayers. Specifically, we compared dialysis and biobeads for OG removal to investigate the influence of these methods on bilayer conformation and polymer rearrangement following detergent removal. There was no significant difference found between method, temperature, or time within each method. Our findings provide insight on how biocompatible polymersome production affects the physical properties of the resulting polymersomes. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016 , 54, 1581–1592     

Biomimetic polymersomes as carriers for hydrophilic quantum dots

For polymersomes to achieve their potential as effective delivery vehicles, they must efficiently encapsulate therapeutic agents into either the aqueous interior or the hydrophobic membrane. In this study, cell membrane-mimetic polymersomes were prepared from amphiphilic poly(D,L-lactide)-b-poly(2-methacryloyloxyethylphosphorylcholine) (PLA-b-PMPC) diblock copolymers and were used as encapsulation devices for water-soluble molecules. Thioalkylated zwitterionic phosphorylcholine protected quantum dots (PC@QDs) were chosen as hydrophilic model substrates and successfully encapsulated into the aqueous polymersome interior, as evidenced by transmission electron microscopy (TEM) and flow cytometry. In addition, we also found a fraction of the PC@QDs were bound to both the external and internal surfaces of the polymersome. This interesting immobilization might be due to the ion-pair interactions between the phosphorylcholine groups on the PC@QDs and polymersomes. The experimental encapsulation results support a mechanism of PLA-b-PMPC polymersome formation in which PLA-b-PMPC copolymer chains first form spherical micelles, then worm-like micelles, and finally disk-like micelles which close up to form polymersomes.     

Polymersome Popping by Light‐Induced Osmotic Shock under Temporal,Spatial, and Spectral Control

The light‐triggered, programmable rupture of cell‐sized vesicles is described, with particular emphasis on self‐assembled polymersome capsules. The mechanism involves a hypotonic osmotic imbalance created by the accumulation of photogenerated species inside the lumen, which cannot be compensated owing to the low water permeability of the membrane. This simple and versatile mechanism can be adapted to a wealth of hydrosoluble molecules, which are either able to generate reactive oxygen species or undergo photocleavage. Ultimately, in a multi‐compartmentalized and cell‐like system, the possibility to selectively burst polymersomes with high specificity and temporal precision and to consequently deliver small encapsulated vesicles (both polymersomes and liposomes) is demonstrated.     

Polymersome Popping by Light‐Induced Osmotic Shock under Temporal,Spatial, and Spectral Control

The light‐triggered, programmable rupture of cell‐sized vesicles is described, with particular emphasis on self‐assembled polymersome capsules. The mechanism involves a hypotonic osmotic imbalance created by the accumulation of photogenerated species inside the lumen, which cannot be compensated owing to the low water permeability of the membrane. This simple and versatile mechanism can be adapted to a wealth of hydrosoluble molecules, which are either able to generate reactive oxygen species or undergo photocleavage. Ultimately, in a multi‐compartmentalized and cell‐like system, the possibility to selectively burst polymersomes with high specificity and temporal precision and to consequently deliver small encapsulated vesicles (both polymersomes and liposomes) is demonstrated.     

In Vitro Drug and Gene Delivery Using Random Cationic Copolymers Forming Stable and pH‐Sensitive Polymersomes

Stimuli‐sensitive polymeric vesicles or polymersomes as self‐assembled colloidal nanocarriers have received paramount importance for their integral role as delivery system for therapeutics and biotherapeutics. This work describes spontaneous polymersome formation at pH 7, as evidenced by surface tension, steady state fluorescence, dynamic light scattering, and microscopic studies, by three hydrophilic random cationic copolymers synthesized using N ,N‐(dimethylamino)ethyl methacrylate (DMAEM) and methoxy poly(ethylene glycol) monomethacrylate in different mole ratios. The results suggest that methoxy poly(ethylene glycol) chains constitute the bilayer membrane of the polymersomes and DMAEM projects toward water constituting the positively charged surface. The polymersomes have been observed to release their encapsulated guest at acidic pH as a result of transformation into polymeric micelles. All these highly biocompatible cationic polymers show successful gene transfection ability as nonviral vector on human cell line with different potential. Thus these polymers prove their utility as a potential delivery system for hydrophilic model drug as well as genetic material.

     

Photoactivated Polymersome Nanomotors: Traversing Biological Barriers

Synthetic nanomotors are appealing delivery vehicles for the dynamic transport of functional cargo. Their translation toward biological applications is limited owing to the use of non‐degradable components. Furthermore, size has been an impediment owing to the importance of achieving nanoscale (ca. 100 nm) dimensions, as opposed to microscale examples that are prevalent. Herein, we present a hybrid nanomotor that can be activated by near‐infrared (NIR)‐irradiation for the triggered delivery of internal cargo and facilitated transport of external agents to the cell. Utilizing biodegradable poly(ethylene glycol)‐b‐poly(d,l ‐lactide) (PEG‐PDLLA) block copolymers, with the two blocks connected via a pH sensitive imine bond, we generate nanoscopic polymersomes that are then modified with a hemispherical gold nanocoat. This Janus morphology allows such hybrid polymersomes to undergoing photothermal motility in response to thermal gradients generated by plasmonic absorbance of NIR irradiation, with velocities ranging up to 6.2±1.10 μm s^?1. These polymersome nanomotors (PNMs) are capable of traversing cellular membranes allowing intracellular delivery of molecular and macromolecular cargo.     

pH-responsive Polymersome Based on PMCP-b-PDPA as a Drug Delivery System to Enhance Cellular Internalization and Intracellular Drug Release

Choline phosphate(CP) as a novel zwitterion possesses specific and excellent properties compared with phosphorylcholine(PC), as well as its polymer, such as poly(2-(methacryloyloxy)ethyl choline phosphate)(PMCP), has been studied extensively due to its unique characteristics of rapid cellular internalization via the special quadrupole interactions with the cell membrane. Recently, we reported a novel PMCP-based drug delivery system to enhance the cellular internalization where the drug was conjugated to the polymer via reversible acylhydrazone bond. Herein, to make full use of this feature of PMCP, we synthesized the diblock copolymer poly(2-(methacryloyloxy)ethyl choline phosphate)-b-poly(2-(diisopropylamino)ethyl methacrylate)(PMCP-b-PDPA), which could self-assemble into polymersomes with hydrophilic PMCP corona and hydrophobic membrane wall in mild conditions when the p H value is ≥ 6.4. It has been found that these polymersomes can be successfully used to load anticancer drug Dox with the loading content of about 11.30 wt%. After the polymersome is rapidly internalized by the cell with the aid of PMCP, the loaded drug can be burst-released in endosomes since PDPA segment is protonated at low p H environment, which renders PDPA to transfer from hydrophobic to hydrophilic,and the subsequent polymersomes collapse thoroughly. Ultimately, the "proton sponge" effect of PDPA chain can further accelerate the Dox to escape from endosome to cytoplasm to exert cytostatic effects. Meanwhile, the cell viability assays showed that the Dox-loaded polymersomes exhibited significant inhibitory effect on tumor cells, indicating its great potential as a targeted intracellular delivery system with high efficiency.     

Polymersomes as protocellular constructs

Despite the rapidly growing amount of knowledge on the structure and function of cells, they remain a distant bottom‐up synthetic target due to their overwhelming complexity. A path to this goal is the development of protocellular systems that approximate one or more aspects of a natural cell. Polymer vesicles, or polymersomes, are an attractive scaffold for protocellular constructs, due to our ability to engineer the polymer membrane and generate a wide range of properties. This article summarizes the current state of polymersome science with respect to the properties and functions that lend these polymer‐based systems to applications in synthetic cell research. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 3817–3825     

Self‐assembly of thermo‐ and pH‐responsive poly(acrylic acid)‐b‐poly(N‐isopropylacrylamide) micelles for drug delivery

Self‐assembled thermo‐ and pH‐responsive poly(acrylic acid)‐b‐poly(N‐isopropylacrylamide) (PAA‐b‐PNIPAM) micelles for entrapment and release of doxorubicin (DOX) was described. Block copolymer PAA‐b‐PNIPAM associated into core‐shell micelles in aqueous solution with collapsed PNIPAM block or protonated PAA block as the core on changing temperature or pH. Complexation of DOX with PAA‐b‐PNIPAM triggered by the electrostatic interaction and release of DOX from the complexes due to the changing of pH or temperature were studied. Complex micelles incorporated with DOX exhibited pH‐responsive and thermoresponsive drug release profile. The release of DOX from micelles was suppressed at pH 7.2 and accelerated at pH 4.0 due to the protonation of carboxyl groups. Furthermore, the cumulative release of DOX from complex micelles was enhanced around LCST ascribed to the structure deformation of the micelles. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 5028–5035, 2008     

A Biodegradable Polymersome Containing Bcl‐xL siRNA and Doxorubicin as a Dual Delivery Vehicle for a Synergistic Anticancer Effect

Combined cancer treatment via co‐delivery of siRNAs and an anticancer drug can be a promising strategy due to the synergistic effect of simultaneously minimizing gene/drug administration. In this study, Bcl‐xL siRNA and doxorubicin (DOX) are encapsulated into designed methoxy‐poly(ethylene glycol)‐block‐poly(D ,L ‐lactic acid) (mPEG‐b‐PLA) block copolymer polymersomes (PSomes). A study of the cytotoxicity of Bcl‐xL siRNA and DOX co‐encapsulated PSomes (CPSomes) shows more inhibited proliferation of MKN‐45 and MKN‐28 human gastric cancer cell lines than only gene‐ and drug‐loaded ones. Consequently, these results demonstrate that co‐delivery of genes and drugs using PSomes results in a synergistic efficacy and indicates the potential of PSomes as efficient nanocarriers for combined cancer therapy.

     

Synthesis and characterization of a biodegradable ABC triblock terpolymer as co‐delivery carrier of doxorubicin and DNA

This study is aimed to develop a well‐defined ABC triblock terpolymer, poly(ethylethylene phosphate)‐block‐poly(ε‐caprolactone)‐block‐poly[2‐(dimethylamino)ethyl methacrylate] (PEEP‐b‐PCL‐b‐PDMAEMA), for co‐encapsulating anticancer drug doxorubicin (DOX) and DNA to form polyplexes. The terpolymer is first synthesized via a combination of ring‐opening polymerization and atom‐transfer radical polymerization techniques, and characterized by ¹H NMR and gel permeation chromatography. Subsequently, the self‐assembly behavior of the terpolymer and the micelles loaded with DOX or DNA are investigated by dynamic light scattering, ζ potential, transmission electron microscopy, and gel retardation assay, respectively. In vitro release study reveals that much more DOX is released at pH 5.0 than that at pH 7.4 in the same period. The simultaneous delivery of DOX and green fluorescent protein (GFP)‐labeled DNA is studied by a fluorescence microscope and the results demonstrate that both drug and GFP–DNA can be efficiently delivered into HeLa cells. This system presents a practical and promising carrier for the co‐delivery of drugs and genes. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 3005–3016     

Controlling Fusion and Aggregation in Polymersome Dispersions

The effects of addition of poly(ethylene oxide) (PEO) to poly(ethylene oxide)₁₆‐block‐poly(butylene oxide)₂₂ (EB1) polymersome dispersions has been studied. Aggregation and membrane fusion between polymersomes were observed with great control of the outcome by varying PEO molecular weight. Small‐scale fusion of 2 to 3 polymersomes was seen with low‐molecular‐weight PEO, whilst large‐scale aggregation occurred above a critical PEO molecular weight of 4 000 Da. The resulting highly porous EB1‐PEO aggregate shows great promise for applications in cell entrapment for transport and delivery, and as a three‐dimensional scaffold for use in tissue engineering.

     

pH-responsive polymersome based on PMCP-<Emphasis Type="Italic">b</Emphasis>-PDPA as a drug delivery system to enhance cellular internalization and intracellular drug release

Choline phosphate (CP) as a novel zwitterion possesses specific and excellent properties compared with phosphorylcholine (PC), as well as its polymer, such as poly(2-(methacryloyloxy)ethyl choline phosphate) (PMCP), has been studied extensively due to its unique characteristics of rapid cellular internalization via the sepcial quadrupole interactions with the cell membrane. Recently, we reported a novel PMCP-based drug delivery system to enhance the cellular internalization where the drug was conjugated to the polymer via reversible acylhydrazone bond. Herein, to make full use of this feature of PMCP, we synthesized the diblock copolymer poly(2-(methacryloyloxy)ethyl choline phosphate)-b-poly(2-(diisopropylamino)ethyl methacrylate) (PMCP-b-PDPA), which could self-assemble into polymersomes with hydrophilic PMCP corona and hydrophobic membrane wall in mild conditions when the pH value is ≥ 6.4. It has been found that these polymersomes can be successfully used to load anticancer drug Dox with the loading content of about 11.30 wt%. After the polymersome is rapidly internalized by the cell with the aid of PMCP, the loaded drug can be burst-released in endosomes since PDPA segment is protonated at low pH environment, which renders PDPA to transfer from hydrophobic to hydrophilic, and the subsequent polymersomes collapse thoroughly. Ultimately, the “proton sponge” effect of PDPA chain can further accelerate the Dox to escape from endosome to cytoplasm to exert cytostatic effects. Meanwhile, the cell viability assays showed that the Dox-loaded polymersomes exhibited significant inhibitory effect on tumor cells, indicating its great potential as a targeted intracellular delivery system with high efficiency.     

Fluorescent Supramolecular Polymersomes Based on Pillararene/Paraquat Molecular Recognition for pH-controlled Drug Release

Researchers have put significant efforts on developing versatile fluorescent polymeric systems due to their promising biological/biomedical labelling, tracking, monitoring, imaging, and diagnostic applications. However, complicated organic/polymeric synthesis or post-modification of these functionalized platforms is still a big obstacle for their further application and thereby provides clear motivation for exploring alternative strategies for the design and fabrication of easily available fluorescent systems. The marriage of supramolecular polymers and fluorescent imaging can provide a facile and dynamic manner instead of tedious and time-consuming synthesis due to the dynamic and reversible nature of noncovalent interactions. Herein, based on water-soluble pillararene/paraquat molecular recognition, we successfully prepare two amphiphilic polypseudorotaxanes which can self-assemble into supramolecular polymersomes in water. These polymersomes can be reversibly destroyed and reformed by tuning the solution p H. Attributed to the aggregation-induced emission of tetraphenylethylene groups,intense fluorescence can be introduced into the obtained supramolecular polymersomes. Furthermore, p H-triggered release of an encapsulated water-insoluble drug(doxorubicin) from the self-assembled fluorescent supramolecular polymersomes is also investigated.     

Temperature‐Induced Transformation from Large Compound Vesicles to Worm‐like Aggregates by ABC Triblock Copolymer

Two triblock polymers, tetraaniline‐block‐poly(N‐isopropyl acrylamide)‐block‐poly(hydroxyethyl acrylate) (TA‐b‐PNIPAM‐b‐PHEA) and TA‐b‐PHEA‐b‐PNIPAM, were synthesized with unambiguous structure by a two step method. The difference of these two diblock polymers is the connection order of carboxyl group to block, e.g., carboxyl group to PNIPAM block for PNIPAM‐b‐PHEA and to PHEA block for PHEA‐b‐PNIPAM. Secondly, block tetraaniline was linked to the diblock polymer through amidation to yield the corresponding triblock copolymer. Both of them have almost the identical chemical compositions. The only difference is the connection order of each block in the triblock polymers. When they were self‐assembled at 45°C in a suitable solution, both of their aggregates have spherical shape with slight defects on their surface with the average diameter of about 400 nm. However, when their aggregate dispersion was cooled down to 20°C, only TA‐b‐PHEA‐b‐PNIPAM's morphology changed, forming worm‐like aggregates with the diameter of about 100–200 nm transformed from spherical aggregates. Both amphiphilic property and position of each block in this triblock copolymer are very essential for this morphology transformation. Since the worm‐like aggregates presented here by our group have hollow structure inside, its controlled release properties for doxorubicin were evaluated. Drug release experiment indicated that along with the temperature changes, the rearrangement of the intermediate layer structure caused morphology change in aggregate, thus accelerating the speed of drug release.