Micelle or polymersome formation by PCL-PEG-PCL copolymers as drug delivery systems

Polymeric micelles and polymersomes may have great potential as the drug delivery vehicles for solubilization of hydrophobic drugs.     

Can polymersomes form colloidosomes?

Hydroxy-functionalized polymersomes (or block copolymer vesicles) were prepared via a facile one-pot RAFT aqueous dispersion polymerization protocol and evaluated as Pickering emulsifiers for the stabilization of emulsions of n-dodecane emulsion droplets in water. Linear polymersomes produced polydisperse oil droplets with diameters of ～50 μm regardless of the polymersome concentration in the aqueous phase. Introducing an oil-soluble polymeric diisocyanate cross-linker into the oil phase prior to homogenization led to block copolymer microcapsules, as expected. However, TEM inspection of these microcapsules after an alcohol challenge revealed no evidence for polymersomes, suggesting these delicate nanostructures do not survive the high-shear emulsification process. Thus the emulsion droplets are stabilized by individual diblock copolymer chains, rather than polymersomes. Cross-linked polymersomes (prepared by the addition of ethylene glycol dimethacrylate as a third comonomer) also formed stable n-dodecane-in-water Pickering emulsions, as judged by optical and fluorescence microscopy. However, in this case the droplet diameter varied from 50 to 250 μm depending on the aqueous polymersome concentration. Moreover, diisocyanate cross-linking at the oil/water interface led to the formation of well-defined colloidosomes, as judged by TEM studies. Thus polymersomes can indeed stabilize colloidosomes, provided that they are sufficiently cross-linked to survive emulsification.     

Glucose-oxidase based self-destructing polymeric vesicles

We have designed oxidation-responsive vesicles from synthetic amphiphilic block copolymers ("polymersomes") of ethylene glycol and propylene sulfide. Thioethers in the hydrophobic poly(propylene sulfide) block are converted into the more hydrophilic sulfoxides and sulfones upon exposure to an oxidative environment, changing the hydrophilic-lipophilic balance of the macroamphiphile and thus inducing its solubilization. Here we sought to explore generation of the oxidative environment and induction of polymersome destabilization through production of hydrogen peroxide by the glucose-oxidase (GOx)/glucose/oxygen system. We studied the encapsulation of GOx within polymersomes, its stability and activity, and glucose-triggered polymersome destabilization. Stimulus-responsive polymersomes may find applications as nanocontainers in sensing devices and as drug delivery systems.     

Microfluidic fabrication of monodisperse biocompatible and biodegradable polymersomes with controlled permeability

We describe a versatile technique for fabricating monodisperse polymersomes with biocompatible and biodegradable diblock copolymers for efficient encapsulation of actives. We use double emulsion as a template for the assembly of amphiphilic diblock copolymers into vesicle structures. These polymersomes can be used to encapsulate small hydrophilic solutes. When triggered by an osmotic shock, the polymersomes break and release the solutes, providing a simple and effective release mechanism. The technique can also be applied to diblock copolymers with different hydrophilic-to-hydrophobic block ratios, or mixtures of diblock copolymers and hydrophobic homopolymers. The ability to make polymer vesicles with copolymers of different block ratios and to incorporate different homopolymers into the polymersomes will allow the tuning of polymersome properties for specific technological applications.     

Biologically active polymersomes from amphiphilic glycopeptides

Polypeptide block copolymers with different block length ratios were obtained by sequential ring-opening polymerization of benzyl-L-glutamate and propargylglycine (PG) N-carboxyanhydrides. Glycosylation of the poly(PG) block was obtained by Huisgens cycloaddition "click" reaction using azide-functionalized galactose. All copolymers were self-assembled using the nanoprecipitation method to obtain spherical and wormlike micelles as well as polymersomes depending on the block length ratio and the nanoprecipitation conditions. These structures display bioactive galactose units in the polymersome shell, as proven by selective lectin binding experiments.     

Polymersomes Prepared from Thermoresponsive Fluorescent Protein–Polymer Bioconjugates: Capture of and Report on Drug and Protein Payloads

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein–polymer bioconjugate PNIPAM‐b‐amilFP497 composed of thermoresponsive poly(N‐isopropylacrylamide) (PNIPAM) and a green‐fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co‐)encapsulate the fluorescent drug doxorubicin and the fluorescent light‐harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM‐FRET), we can distinguish the co‐encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.     

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.     

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.     

Enhancement of cellular uptake and antitumor efficiencies of micelles with phosphorylcholine

Internalization of drug delivery micelles into cancer cells is a crucial step for antitumor therapeutics. Novel amphiphilic star-shaped copolymers with zwitterionic phosphorylcholine (PC) block, 6-arm star poly(ε-caprolactone)-b-poly(2-methacryloyloxyethyl phosphorylcholine) (6sPCL-b-PMPC), have been developed for encapsulation of poorly water-soluble drugs and enhancement of their cellular uptake. The star-shaped copolymers were synthesized by a combination of ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP). The copolymers self-assembled to form spherical micelles with low critical micelle concentration (CMC). The sizes of the micelles range from 80 to 170 nm and increase 30 ≈ 80% after paclitaxel (PTX) loading. Labeled with fluorescein isothiocyanate (FITC), the micelles were confirmed by fluorescence microscopy to have been internalized efficiently by tumor cells. Direct visualization of the micelles within tumor cells by transmission electron microscopy (TEM) confirmed that the 6sPCL-b-PMPC micelles were more efficiently uptaken by tumor cells compared to PCL-b-PEG micelles. When incorporated with PTX, the 6sPCL-b-PMPC micelles show much higher cytotoxicity against Hela cells than PCL-b-PEG micelles, in response to the higher efficiency of cellular uptake.     

Multiple polymersomes for programmed release of multiple components

Long-term storage and controlled release of multiple components while avoiding cross-contamination have potentially important applications for pharmaceuticals and cosmetics. Polymersomes are very promising delivery vehicles but cannot be used to encapsulate multiple independent components and release them in a controlled manner. Here, we report a microfluidic approach to produce multiple polymersomes, or polymersomes-in-polymersome by design, enabling encapsulation and programmed release of multiple components. Monodisperse polymersomes are prepared from templates of double-emulsion drops, which in turn are injected as the innermost phase to form the second level of double-emulsion drops, producing double polymersomes. Using the same strategy, higher-order polymersomes are also prepared. In addition, incorporation of hydrophobic homopolymer into the different bilayers of the multiple polymersomes enables controlled and sequential dissociation of the different bilayer membranes in a programmed fashion. The high encapsulation efficiency of this microfluidic approach, as well as its programmability and the biocompatibility of the materials used to form the polymersomes, will provide new opportunities for practical delivery systems of multiple components.     

Porphyrin Containing Polymersomes with Enhanced ROS Generation Efficiency: In Vitro Evaluation

Porphyrins are molecules possessing unique photophysical properties making them suitable for application in photodynamic therapy. The incorporation of porphyrins into natural or synthetic nano‐assemblies such as polymersomes is a strategy to improve and prolong their therapeutic capacities and to overcome their limitations as therapeutic and diagnostic agents. Here, 5,10,15,20‐tetrakis(1‐(6‐ethoxy‐6‐oxohexyl)‐4‐pyridin‐1‐io)‐21H,23H‐porphyrin tetrabromide porphyrin is inserted into polymersomes in order to demonstrate that the encapsulation enhances its ability to generate highly reactive singlet oxygen (¹O₂) upon irradiation in vitro. The photoactivation of the free and polymersome‐encapsulated porphyrin is evaluated by electron spin resonance and cell viability assays on three different mammalian cell lines. The results indicate that by encapsulating the porphyrin, a controlled ROS delivery within the cells is achieved, at the same time avoiding side effects such as dark toxicity, non‐specific porphyrin release and over time decreased activity in vitro. This work focuses on showing a not‐toxic model system for modern therapeutic nanomedicine, which works under mild irradiation and dosage conditions.     

Polymersome-Embedded Nanoparticles

Summary: Polymersomes are self assembled vesicles composed of fully synthetic amphiphilic diblock copolymers. Many of their properties are similar to lipid vesicles, although with often higher thermal and mechanical stability within their curved membrane. Incorporation of nanosized objects into their hydrophilic interior or hydrophobic membrane represents an important method for functionalization of these biomimetic structures. We report on the embedding of hydrophobic gold nanoparticles (Au-NPs) into polymersomes. Nanoparticles were prepared and incorporated into polymersomes made by standard film rehydration techniques from commercially available diblock copolymers. Characterization of the resulting structures was achieved by dynamic light scattering (DLS) and by TEM. Results on the preparation method, its influence on the polymersome stability and the application of these new membrane-mimetics are reported.     

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.     

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.     

Polymer vesicles with a colloidal armor of nanoparticles

The fabrication of polymer vesicles with a colloidal armor made from a variety of nanoparticles is demonstrated. In addition, it is shown that the armored supracolloidal structure can be postmodified through film-formation of soft polymer latex particles on the surface of the polymersome, hereby effectively wrapping the polymersome in a plastic bag, as well as through formation of a hydrogel by disintegrating an assembled polymer latex made from poly(ethyl acrylate-co-methacrylic acid) upon increasing the pH. Furthermore, ordering and packing patterns are briefly addressed with the aid of Monte Carlo simulations, including patterns observed when polymersomes are exposed to a binary mixture of colloids of different size.     

Photodynamic Effects of Zinc(II) Phthalocyanine‐Loaded Polymeric Micelles in Human Nasopharynx KB Carcinoma Cells

A major difficulty in photodynamic therapy is the poor solubility of the photosensitizer (PS) under physiological conditions which correlates with low bioavailability. PS aggregation leads to a decrease in the photodynamic efficiency and a more limited activity in vitro and in vivo. To improve the aqueous solubility and reduce the aggregation of 2,9(10),16(17),23(24)‐tetrakis[(2‐dimethylamino)ethylsulfanyl]phthal‐ocyaninatozinc(II) (Pc9), the encapsulation into four poloxamine polymeric micelles (T304, T904, T1107 and T1307) displaying a broad spectrum of molecular weight and hydrophilic–lipophilic balance was investigated. The aqueous solubility of Pc9 was increased up to 30 times. Morphological evaluation showed the formation of Pc9‐loaded spherical micelles in the nanosize range. UV/Vis and fluorescence studies indicated that Pc9 is less aggregated upon encapsulation in comparison with Pc9 in water–DMSO 2% and remained photostable. Pc9‐loaded micelles generated singlet molecular oxygen in high yields. Photocytotoxicity assays using human nasopharynx KB carcinoma cells confirmed that the encapsulation of Pc9 in T1107 and T1307 increases its photocytotoxicity by 10 times in comparison with the free form in water–DMSO. In addition, Pc9 incorporated into cells was mainly localized in lysosomes.     

Micellization and reversible pH-sensitive phase transfer of the hyperbranched multiarm PEI-PBLG Copolymer

A novel, hyperbranched, amphiphilic multiarm biodegradable polyethylenimine-poly(gamma-benzyl-L-glutamate) (PEI-PBLG) copolymer was prepared by the ring-opening polymerization of gamma-benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) with hyperbranched PEI as a macroinitiator. The copolymer could self-assemble into core-shell micelles in aqueous solution with highly hydrophobic micelle cores. As the PBLG content was increased, the size of the micelles increased and the critical micelle concentration (CMC) decreased. The surface of the micelles had a positive zeta potential. The cationic micelles were capable of complexing with plasmid DNA (pDNA), which could be released subsequently by treatment with polyanions. The PEI-PBLG copolymer formed unimolecular micelles in chloroform solution. The pH-sensitive phase-transfer behavior exhibited two critical pH points for triggering the encapsulation and release of guest molecules. Both the encapsulation and release processes were rapid and reversible. Under strong acidic or alkaline conditions, the release process became partially or completely irreversible. Thus, this copolymer system should be an attractive candidate for a gene- or drug-delivery system in aqueous media and could provide the phase-transfer carriers between water and organic media.     

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.     

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.