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.     

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.     

Formation of polymer vesicles by liquid crystal amphiphilic block copolymers

We report the formation of polymer vesicles (or polymersomes) by a new class of amphiphilic block copolymers in which the hydrophobic block is a side-on nematic liquid crystal polymer. Two series of these block copolymers, named PEG-b-PA444 and PEG-b-PMAazo444, with different hydrophilic/hydrophobic ratios were synthesized and characterized in detail. Polymersomes and nanotubes were formed by adding water into a solution of copolymers in dioxane. Polymersomes in water were finally obtained by dialyzing the resulting mixture against water. These self-assemblies have been studied by classical TEM and cryo-TEM. For the PEG-b-PA444 series, polymersomes were observed for hydrophilic/hydrophobic ratios ranging from 40/60 to 19/81. For PEG-b-PMAazo444 series, polymersomes were observed for hydrophilic/hydrophobic ratios ranging from 26/74 to 18/82. For a PEG-b-PA444 sample with hydrophilic/hydrophobic ratio equal to 25/75, a tubular morphology with tube diameter of typically 100 nm and tube length of up to 10 mum was also observed together with polymersomes during addition of water into the polymer solution in dioxane.     

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.     

Monosaccharide-responsive release of insulin from polymersomes of polyboroxole block copolymers at neutral pH

We synthesized a boroxole-containing styrenic monomer that can be polymerized by the reversible addition-fragmentation and chain transfer (RAFT) method. Poly(styreneboroxole) (PBOx) and its block copolymers with a poly(ethylene glycol) (PEG) as a hydrophilic block displayed binding to monosaccharides in phosphate buffer at neutral pH, as quantified by Wang's competitive binding experiments. By virtue of a controlled radical polymerization, we were able to adjust the degree of polymerization of the PBOx block to yield sugar-responsive block copolymers that self-assembled into a variety of nanostructures including spherical and cylindrical micelles and polymer vesicles (polymersomes). Polymersomes of these block copolymers exhibited monosaccharide-responsive disassembly in a neutral-pH medium. We demonstrated the possibility of using these polymersomes as sugar-responsive delivery vehicles for insulin in neutral phosphate buffer (pH 7.4). Encapsulated insulin could be released from the polymersomes only in the presence of sugars under physiologically relevant pH conditions.     

Self-Assembled Plasmonic Vesicles of SERS-Encoded Amphiphilic Gold Nanoparticles for Cancer Cell Targeting and Traceable Intracellular Drug Delivery

We report the development of bioconjugated plasmonic vesicles assembled from SERS-encoded amphiphilic gold nanoparticles for cancer-targeted drug delivery. This new type of plasmonic assemblies with a hollow cavity can play multifunctional roles as delivery carriers for anticancer drugs and SERS-active plasmonic imaging probes to specifically label targeted cancer cells and monitor intracellular drug delivery. We have shown that the pH-responsive disassembly of the plasmonic vesicle, stimulated by the hydrophobic-to-hydrophilic transition of the hydrophobic brushes in acidic intracellular compartments, allows for triggered intracellular drug release. Because self-assembled plasmonic vesicles exhibit significantly different plasmonic properties and greatly enhanced SERS intensity in comparison with single gold nanoparticles due to strong interparticle plasmonic coupling, disassembly of the vesicles in endocytic compartments leads to dramatic changes in scattering properties and SERS signals, which can serve as independent feedback mechanisms to signal cargo release from the vesicles. The unique structural and optical properties of the plasmonic vesicle have made it a promising platform for targeted combination therapy and theranostic applications by taking advantage of recent advances in gold nanostructure based in vivo bioimaging and photothermal therapy and their loading capacity for both hydrophilic (nucleic acids and proteins) and hydrophobic (small molecules) therapeutic agents.     

CO2‐Activated Reversible Transition between Polymersomes and Micelles with AIE Fluorescence

Fluorescent polymersomes with both aggregation‐induced emission (AIE) and CO₂‐responsive properties were developed from amphiphilic block copolymer PEG‐b‐P(DEAEMA‐co‐TPEMA) in which the hydrophobic block was a copolymer made of tetraphenylethene functionalized methacrylate (TPEMA) and 2‐(diethylamino)ethyl methacrylate (DEAEMA) with unspecified sequence arrangement. Four block copolymers with different DEAEMA/TPEMA and hydrophilic/hydrophobic ratios were synthesized, and bright AIE polymersomes were prepared by nanoprecipitation in THF/water and dioxane/water systems. Polymersomes of PEG₄₅‐b‐P(DEAEMA₃₆‐co‐TPEMA₆) were chosen to study the CO₂‐responsive property. Upon CO₂ bubbling vesicles transformed to small spherical micelles, and upon Ar bubbling micelles returned to vesicles with the presence of a few intermediate morphologies. These polymersomes might have promising applications as sensors, nanoreactors, or controlled release systems.     

Filling Polymersomes with Polymers by Peroxidase‐Catalyzed Atom Transfer Radical Polymerization

Polymersomes that encapsulate a hydrophilic polymer are prepared by conducting biocatalytic atom transfer radical polymerization (ATRP) in these hollow nanostructures. To this end, ATRPase horseradish peroxidase (HRP) is encapsulated into vesicles self‐assembled from poly(dimethylsiloxane)‐block‐poly(2‐methyl‐2‐oxazoline) (PDMS‐b‐PMOXA) diblock copolymers. The vesicles are turned into nanoreactors by UV‐induced permeabilization with a hydroxyalkyl phenone and used to polymerize poly(ethylene glycol) methyl ether acrylate (PEGA) by enzyme‐catalyzed ATRP. As the membrane of the polymersomes is only permeable for the reagents of ATRP but not for macromolecules, the polymerization occurs inside of the vesicles and fills the polymersomes with poly(PEGA), as evidenced by ¹H NMR. Dynamic and static light scattering show that the vesicles transform from hollow spheres to filled spheres during polymerization. Transmission electron microscopy (TEM) and cryo‐TEM imaging reveal that the polymersomes are stable under the reaction conditions. The polymer‐filled nanoreactors mimic the membrane and cytosol of cells and can be useful tools to study enzymatic behavior in crowded macromolecular environments.

     

PEGylated liposomal doxorubicin targeted to α5β1-expressing MDA-MB-231 breast cancer cells

Targeting drugs selectively to cancer cells can potentially benefit cancer patients by avoiding side effects generally associated with several cancer therapies. One of the attractive approaches to direct the drug cargo to specific sites is to incorporate ligands at the surface of the delivery systems. Integrin α(5)β(1) is overexpressed in tumor vasculature and cancer cells, thus making it an attractive target for use in drug delivery. Our group has developed a fibronectin-mimetic peptide, PR_b, which has been shown to bind specifically to integrin α(5)β(1), thereby providing a tool to target α(5)β(1)-expressing cancer cells in vitro as well as in vivo. Our current work focuses on designing modified stealth liposomes (liposomes functionalized with polyethylene glycol, PEG) for combining the benefits associated with PEGylation, as well as imparting specific targeting properties to the liposomes. We have designed PEGylated liposomes that incorporate in their bilayer the fibronectin-mimetic peptide-amphiphile PR_b that can target several cancer cells that overexpress α(5)β(1), including the MDA-MB-231 breast cancer cells used in this study. We have encapsulated doxorubicin inside the liposomes to enhance its therapeutic potential via PEGylation as well as active targeting to the cancer cells. Our results show that PR_b-functionalized stealth liposomes were able to specifically bind to MDA-MB-231 cells, and the binding could be controlled by varying the peptide concentration. The intracellular trafficking of the doxorubicin liposomes was examined, and within minutes after delivery the majority of them were found to be in the early endosomes, whereas after a longer period of time they had accumulated in the late endosomes and lysosomes. The functionalized liposomes were found to be equally cytotoxic as the free doxorubicin, especially at higher doxorubicin concentrations, and provided higher cytotoxicity than the nontargeted and GRGDSP-functionalized stealth liposomes. Thus, the PR_b-functionalized PEGylated nanoparticles examined in this study offer a promising strategy to deliver their therapeutic payload directly to the breast cancer cells, in an efficient and specific manner.     

PR_b-targeted PEGylated liposomes for prostate cancer therapy

In recent years, there has been considerable effort in designing improved delivery systems by including site-directed surface ligands to further enhance their selective targeting. The goal of this study is to engineer alpha5beta1-targeted stealth liposomes (nanoparticles covered with poly(ethylene glycol) (PEG)) that will bind to alpha5beta1-expressing LNCaP human prostate cancer cells and efficiently release the encapsulated load intracellularly. For this purpose, liposomes (with and without PEG2000) were functionalized with a fibronectin-mimetic peptide (PR_b) and delivered to LNCaPs. The amount of PEG2000 and other liposomal components were characterized by 1H NMR, and the amount of peptide by the bicinchoninic acid protein assay. Fibronectin is the natural ligand for alpha5beta1, and a promising design for a fibronectinmimetic peptide includes both the primary binding site (RGD) and the synergy site (PHSRN) connected by a linker and extended off a surface by a spacer. We have previously designed a peptide-amphiphile, PRb, that employed a hydrophobic tail, connected to the N-terminus of a peptide headgroup composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. We have examined different liposomal formulations, functionalized only with PR_b or with PR_b and PEG2000. For PR_b-targeted PEGylated liposomes, efficient cell binding was observed for peptide concentrations of 2 mol % and higher. When compared to GRGDSP-targeted stealth liposomes, PR_b functionalization was superior to that of GRGDSP as shown by increased LNCaP binding, internalization efficiency, as well as cytotoxicity after incubation of LNCaPs with tumor necrosis factor-alpha (TNFalpha)-encapsulated liposomes. More importantly, PR_b is alpha5beta1-specific, whereas many integrins bind to small RGD peptides. Thus, the proposed PR_b-targeted delivery system has the potential to deliver a therapeutic payload to prostate cancer cells in an efficient and specific manner.     

Polymer vesicles as microreactors for bioinspired calcium carbonate precipitation

Giant polymer vesicles made by electroformation have been shown to encapsulate salts up to concentrations of about 10 mM. The impermeability of these "polymersomes" to calcium ions is demonstrated by the use of fluorescent probes dedicated to calcium analysis. Permeability to calcium ions can be triggered by the addition of calcimycin, an ionophore molecule that is able to transport cations selectively through the membrane. As a result, we show that the mineralization of calcium carbonate can be induced within the polymersomes, which were previously loaded with carbonate ions. This is a further step toward the use of polymersomes as microreactors and the study of mineralization schemes, including biomimetic ones, in confined environments.     

Adhesion of antibody-functionalized polymersomes

Polymersomes are vesicles made from synthetic block copolymers. The adhesiveness of micron-sized polymersomes, functionalized with antibodies that bind to vascular cell adhesion molecules, which could be useful for vascular targeting, was measured. Intercellular adhesion molecule-1 (ICAM-1) is an endothelial cell adhesion molecule whose expression increases during inflammatory disease, and is therefore a natural target for vascular delivery. We functionalized polymersomes with an anti-ICAM-1 antibody, using modular biotin-avidin chemistry. Micropipet aspiration was used to confirm specific adhesion and measure the adhesion strength between an anti-ICAM-1-coated polymersome and an ICAM-1-coated polystyrene microsphere at various surface densities of adhesion molecules. The adhesion is kinetically trapped, and adhesion strength is quantified by the critical tension for detachment. The adhesion strength increases in proportion to the surface density of anti-ICAM-1 molecules, in contrast to results seen previously when measuring adhesion between biotinylated vesicles and avidin-coated beads (Lin et al. Langmuir 2004, 20, 5493). The difference in dependence on the density of functional groups is likely due to the molecular presentation at the vesicle surface; in the current study, the presentation of biotinylated anti-ICAM-1 on a layer of avidin leads to the effective presentation of the anti-ICAM-1 and, thus, a monotonic increase in adhesiveness with antibody density.     

Reversibly self-assembled pH-responsive PEG-p(CL-g-TMC) polymersomes

Polymersomes have gained much interest within the biomedical field as drug delivery systems due to their ability to transport and protect cargo from the harsh environment inside the body. For an improved drug efficacy, control over cargo release is however also an important factor to take into account. An often employed method is to incorporate pH sensitive groups in the vesicle membrane, which induce disassembly and content release when the particles have reached a target site in the body with the appropriate pH, such as the acidic microenvironment of tumor tissue or the endosome. In this paper, biodegradable poly(ethylene glycol)-poly(caprolactone-gradient-trimethylene carbonate)-based polymeric vesicles have been developed with disassembly features at mild acidic conditions. Modifying the polymer backbone with imidazole moieties results in vesicle disassembly upon protonation due to the lowered pH. Furthermore, upon increasing the pH efficient re-assembly into vesicles is observed due to the switchable amphiphilic nature of the polymer. When this re-assembly process is conducted in presence of cargo, enhanced encapsulation is achieved. Furthermore, the potency of the polymeric system for future biomedical applications such as adjuvant delivery is demonstrated.     

How does the hydrophobic content of methacrylate ABA triblock copolymers affect polymersome formation?

Polymersomes are exciting self-assembled structures with great potential in pharmaceutical applications. A systematic investigation of a novel series of methacrylate-based polymersomes is reported in this study. Five well-defined ABA triblock copolymers with A being based on tri(ethylene glycol) methyl methacrylate and B being based on 2-(diethylamino)ethyl methacrylate (DMAEMA) were synthesized using a living polymerization method. The effect of the composition of the ABA triblock copolymers on the thickness of the hydrophobic membrane of the polymersomes and the release of a model drug is demonstrated.     

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.     

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.     

Photo-crosslinked and pH sensitive polymersomes for triggering the loading and release of cargo

Crosslinkable and pH-sensitive amphiphilic block copolymers are promising candidates to establish pH-stable and permeable vesicles for synthetic biology. Here, we report the fabrication of crosslinked and pH-stable polymersomes as swellable vesicles for the pH-dependent loading and release of small dye molecules.     

Target delivery of photo-triggered nanocarrier for externally activated chemo-photodynamic therapy of prostate cancer

Objective therapeutics such as photodynamic therapy (PDT) play an imperative role where targeted delivery of nanotherapeutics could achieve the highest level of therapeutic efficiency for the treatment of cancer. For an effective combination of chemotherapy and PDT, a multimodal-targeted system is vital to achieving effective therapeutic efficacy to counter cancer. In this study, an upconversion nanoparticle-based dual-mode nanocarrier was established where doxorubicin, a chemotherapeutic drug, and tetra carboxy zinc phthalocyanine, a reactive oxygen species (ROS) generator, were successfully embedded onto metal-organic framework (ZIF-8) for synergistic photodynamic therapy. For controlled drug release, amine-PEG was wrapped around UCNPs@MOF. In addition, targeting efficiency was enhanced by employing a prostate cancer-specific ligand (folic acid, FA), which is recognized by prostate-specific membrane antigen (PSMA). Indeed, the nanocomposite-coupled FA was uptaken more in LNCaP (PSMA positive) cells compared to DU145 (PSMA negative) cells. Interestingly, coating the nanocomposite with biocompatible polyethylene glycol significantly inhibited doxorubicin (DOX) release even under a lower pH condition. This effect is abrogated by near-infrared irradiation, whereupon NIR irradiation, the nanocomposite accelerates the production of ROS, as well as chemotherapeutic drug release. These results suggest that the release of DOX was more tightly controlled by a polymer coating. As observed by in vitro cytotoxicity experiment, LNCaP cells showed descending pattern in the cell viability than DU145 cells under the NIR irradiation condition. All these results, taken together, show a promising system for NIR-based targeted PDT where burst release of drug and ROS is achieved to improve the synergistic therapeutics.     

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.     

Engineering Polymersome Protocells

The field of biomimicry is embracing the construction of complex assemblies that imitate both biological structure and function. Advancements in the design of these mimetics have generated a growing vision for creating an artificial or proto- cell. Polymersomes are vesicles that can be made from synthetic, biological or hybrid polymers and can be used as a model template to build cell-like structures. In this perspective, we discuss various areas where polymersomes have been used to mimic cell functions as well as areas in which the synthetic flexibility of polymersomes would make them ideal candidates for a biomembrane mimetic. Designing a polymersome that comprehensively displays the behaviors discussed herein has the potential to lead to the development of an autonomous, responsive particle that resembles the intelligence of a biological cell.