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.     

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.     

Targeted Polymersome Delivery of siRNA Induces Cell Death of Breast Cancer Cells Dependent upon Orai3 Protein Expression

Polymersomes, polymeric vesicles that self-assemble in aqueous solutions from block copolymers, have been avidly investigated in recent years as potential drug delivery agents. Past work has highlighted peptide-functionalized polymersomes as a highly promising targeted delivery system. However, few reports have investigated the ability of polymersomes to operate as gene delivery agents. In this study, we report on the encapsulation and delivery of siRNA inside of peptide-functionalized polymersomes composed of poly(1,2-butadiene)-b-poly(ethylene oxide). In particular, PR_b peptide-functionalized polymer vesicles are shown to be a promising system for siRNA delivery. PR_b is a fibronectin mimetic peptide targeting specifically the α(5)β(1) integrin. The Orai3 gene was targeted for siRNA knockdown, and PR_b-functionalized polymer vesicles encapsulating siRNA were found to specifically decrease cell viability of T47D breast cancer cells to a certain extent, while preserving viability of noncancerous MCF10A breast cells. siRNA delivery by PR_b-functionalized polymer vesicles was compared to that of a current commercial siRNA transfection agent, and produced less dramatic decreases in cancer cell viability, but compared favorably in regards to the relative toxicity of the delivery systems. Finally, delivery and vesicle release of a fluorescent encapsulate by PR_b-functionalized polymer vesicles was visualized by confocal microscopy, and colocalization with cellular endosomes and lysosomes was assessed by organelle staining. Polymersomes were observed to primarily release their encapsulate in the early endosomal intracellular compartments, and data may suggest some escape to the cytosol. These results represent a promising first generation model system for targeted delivery of siRNA.     

A Three-Enzyme Cascade Reaction through Positional Assembly of Enzymes in a Polymersome Nanoreactor

Porous polymersomes based on block copolymers of isocyanopeptides and styrene have been used to anchor enzymes at three different locations, namely, in their lumen (glucose oxidase, GOx), in their bilayer membrane (Candida antarctica lipase B, CalB) and on their surface (horseradish peroxidase, HRP). The surface coupling was achieved by click chemistry between acetylene-functionalised anchors on the surface of the polymersomes and azido functions of HRP, which were introduced by using a direct diazo transfer reaction to lysine residues of the enzyme. To determine the encapsulation and conjugation efficiency of the enzymes, they were decorated with metal-ion labels and analysed by mass spectrometry. This revealed an almost quantitative immobilisation efficiency of HRP on the surface of the polymersomes and a more than statistical incorporation efficiency for CalB in the membrane and for GOx in the aqueous compartment. The enzyme-decorated polymersomes were studied as nanoreactors in which glucose acetate was converted by CalB to glucose, which was oxidised by GOx to gluconolactone in a second step. The hydrogen peroxide produced was used by HRP to oxidise 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to ABTS^.+. Kinetic analysis revealed that the reaction step catalysed by HRP is the fastest in the cascade reaction.     

pH and reduction dual-bioresponsive polymersomes for efficient intracellular protein delivery

pH and reduction dual-bioresponsive nanosized polymersomes based on poly(ethylene glycol)-SS-poly(2-(diethyl amino)ethyl methacrylate) (PEG-SS-PDEA) diblock copolymers were developed for efficient encapsulation and triggered intracellular release of proteins. PEG-SS-PDEA copolymers with PDEA-block molecular weights ranging from 4.7, 6.8, to 9.2 kg/mol were synthesized in a controlled manner via reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-(diethyl amino)ethyl methacrylate (DEAEMA) using PEG-SS-CPADN (CPADN = 4-cyanopentanoic acid dithionaphthalenoate; M(n) PEG = 1.9 kg/mol) as a macro-RAFT agent. These copolymers existed as unimers in water at mildly acidic pH (<7.2) conditions, but readily formed monodisperse nanosized polymersomes (54.5-66.8 nm) when adjusting solution pH to 7.4. These polymersomes were highly sensitive to intracellular pH and reductive environments, which resulted in fast dissociation and aggregation of polymersomes, respectively. Notably, both fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (FITC-BSA) and cytochrome C (FITC-CC) proteins could facilely be encapsulated into polymersomes with excellent protein-loading efficiencies, likely as a result of electrostatic interactions between proteins and PDEA. The in vitro release studies showed that protein release was minimal (<20% in 8 h) at pH 7.4 and 37 °C. The release of proteins was significantly enhanced at pH 6.0 due to collapse of polymersomes. Notably, the fastest protein release was observed under intracellular-mimicking reductive environments (10 mM dithiothreitol, pH 7.4). MTT assays in RAW 264.7 and MCF-7 cells indicated that PEG-SS-PDEA (9.2 k) polymersomes had low cytotoxicity up to a polymer concentration of 300 μg/mL. Confocal laser scanning microscope (CLSM) observations revealed that FITC-CC-loaded PEG-SS-PDEA (9.2 k) polymersomes efficiently delivered and released proteins into MCF-7 cells following 6 h of incubation. Importantly, flow cytometry assays showed that CC-loaded PEG-SS-PDEA (9.2 k) polymersomes induced markedly enhanced apoptosis of MCF-7 cells as compared to free CC and CC-loaded PEG-PDEA (8.9 k) polymersomes (reduction-insensitive control). These dual-bioresponsive polymersomes have appeared to be highly promising for intracellular delivery of protein drugs.     

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.     

Towards Targeted Drug Delivery by Covalent Ligand-Modified Polymeric Nanocontainers

Here we present a novel strategy for specific cellular targeting of polymeric nanocontainers by using self-assembly of block copolymers consisting of either Polydimethoxysiloxane-b-Polymethyloxazoline-b-Polydimethoxysiloxane (PDMS-b-PMOXA-b-PDMS) or functionalized PDMS-b-PMOXA-b-PDMS. Covalent functionalization of the above copolymer was accomplished using either the fluorescent dye sulforhodamine B or a poly-guanosin ligand, the latter by using the Huisgen 1,3-dipolar cycloaddition. The success of the covalent modification of the block copolymer has been determined by studying functionalized sulforhodamine B by NMR and fluorescence correlation spectroscopy. The covalent click chemistry approach leads to efficiently functionalized polymeric nanocontainers which enables specific uptake by activated macrophages overexpressing the scavenger receptor A1.     

The effect of polymer chain length and surface density on the adhesiveness of functionalized polymersomes

Giant cell-like polymer vesicles, polymersomes, made from the diblock copolymer poly(ethylene oxide)-polybutadiene (PEO-PBD), have bilayer structures similar to the cell membrane but have superior and tunable properties for storage and stability. We have modified the terminal hydroxyl of the hydrophilic block with biotin-lysine (biocytin), a biologically derived group that imparts specific adhesiveness to a polymer colloid coated with avidin. The functionalized polymer will form vesicles, either on its own or when mixed with unmodified block copolymers that also form vesicles. The incorporation and mixing of the functionalized polymer into vesicle bilayers is measured using a fluorescent version ofbiocytin with confocal microscopy. The fluorescence signal associated with the vesicle is in proportion with the concentration of functional polymer added during vesicle construction. The adhesiveness of polymer vesicles containing functionalized biotinylated polymer to avidin coated microspheres is measured with micropipet aspiration. Two types of polymer vesicles were constructed: one where the functionalized polymer (molecular weight (MW), 10400 Da) was longer than the surrounding unfunctionalized polymer (MW, 3600 Da) and one where the functionalized polymer (MW, 10400 Da) was the same length as the unfunctionalized polymer. In all cases, the avidin-biotin bonds form kinetically trapped crossbridges that impart little tension as they form but require significantly more tension to break. The relative length of the functionalized polymer on the surface of the vesicle is an important determinant for the adhesion of a polymer vesicle but not for the adsorption of soluble avidin. Greater adhesion strengths are seen where the functionalized polymer is longer than the surrounding polymer. The concentration of functionalized polymer at which adhesion is maximal depends on the relative lengths of the polymers. When the functionalized polymer is the same length as the surface brush of the polymersome membrane, the critical tension is maximal at 10 mol % functionalized polymer concentration. However, when the biocytin groups are attached to a polymer which is larger than the surface brush, the critical tension is maximal at 55 mol % functionalized polymer. These results indicate that polymer mixing and length can control the interfacial adhesion of polymer brushes and must be understood to tune polymersome adhesiveness.     

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.     

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.     

Beta-cyclodextrin-appended giant amphiphile: aggregation to vesicle polymersomes and immobilisation of enzymes

A giant amphiphile consisting of polystyrene end-capped with permethylated beta-cyclodextrin was synthesised and found to form vesicular structures when injected as a solution in THF into water. The ability of the cyclodextrins on the surface of the polymersomes to form inclusion complexes with hydrophobic compounds was tested by carrying out a competition experiment with a fluorescent probe sensitive to the polarity of the surrounding medium. It was found that 1-adamantol can displace the fluorescent probe from the cavities of the cyclodextrin moieties of the polymersomes. The recognition of molecules by cell membranes in nature is often based on interactions with specific membrane receptors. To mimic this behaviour, the enzyme horseradish peroxidase was modified with adamantane groups through a poly(ethylene glycol) spacer and its interaction with the polymersomes was investigated. It was established that the presence of adamantane moieties on each enzyme allowed a host-guest interaction with the multifunctional surface of the polymersomes.     

Dewetting instability during the formation of polymersomes from block-copolymer-stabilized double emulsions

We investigate the formation of polymer vesicles, or polymersomes, of polystyrene-block-poly(ethylene oxide) diblock copolymers using double emulsion droplets of controlled architecture as templates. To engineer the structure of the polymersomes, it is important to consider the concentration of diblock copolymer in the middle phase of the double emulsion. We describe how the presence of excess polymer can induce a transition from complete wetting to partial wetting of the middle phase, resulting in polymer shells with inhomogeneous thicknesses.     

Mesoporous silica nanoparticles for 19F magnetic resonance imaging,fluorescence imaging,and drug delivery

Multifunctional mesoporous silica nanoparticles (MSNs) are good candidates for multimodal applications in drug delivery, bioimaging, and cell targeting. In particular, controlled release of drugs from MSN pores constitutes one of the superior features of MSNs. In this study, a novel drug delivery carrier based on MSNs, which encapsulated highly sensitive ¹⁹F magnetic resonance imaging (MRI) contrast agents inside MSNs, was developed. The nanoparticles were labeled with fluorescent dyes and functionalized with small molecule-based ligands for active targeting. This drug delivery system facilitated the monitoring of the biodistribution of the drug carrier by dual modal imaging (NIR/¹⁹F MRI). Furthermore, we demonstrated targeted drug delivery and cellular imaging by the conjugation of nanoparticles with folic acid. An anticancer drug (doxorubicin, DOX) was loaded in the pores of folate-functionalized MSNs for intracellular drug delivery. The release rates of DOX from the nanoparticles increased under acidic conditions, and were favorable for controlled drug release to cancer cells. Our results suggested that MSNs may serve as promising ¹⁹F MRI-traceable drug carriers for application in cancer therapy and bio-imaging.     

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.     

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.     

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.     

Dual silane surface functionalization for the selective attachment of human neuronal cells to porous silicon

Porous silicon (pSi) surfaces were chemically micropatterned through a combination of photolithography and surface silanization reactions. This patterning technique produces discretely defined regions on a pSi surface functionalized with a specific chemical functionality, and the surrounding surface displays a completely different functionality. The generated chemical patterns were characterized by a combination of IR microscopy and the conjugation of two different fluorescent organic dyes. Finally, the chemically patterned pSi surface was used to direct the attachment of neuronal cells to the surface. This patterning strategy will be useful for the development of high-throughput platforms for investigating cell behavior.     

Biotin functionalized poly(sulfonic acid)s for bioconjugation: In situ binding monitoring by QCM‐D

We describe the synthesis of biotin end functionalized poly(sulfonic acid)s via living radical polymerization (LRP) for conjugation to Avidin. Quartz crystal microbalance (QCM‐D) and competitive binding studies were used to confirm this conjugation. A biotin initiator for copper‐mediated LRP was used to provide acrylamide and methacrylate based polymers with the functional end group. This investigation revealed that 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid was not a suitable monomer in its acid form but was successfully used in its sodium salt form. A second monomer, 3‐sulfopropylmethacrylate as the potassium salt was also studied and both monomers produced polymers with polydispersities <1.3 and 1.4, respectively. Evolution of molecular weight with respect to time indicated that the polymerization of the acrylamide polymer is controlled. Quartz crystal microbalance with dissipation monitoring was used to confirm that the biotinylated polymers were able to bind to Avidin in situ. The gold surface of a quartz crystal was chemically modified resulting in a stable monolayer of Avidin; the biotinylated polymers were passed over the functionalized surface and their grafting ability was examined. A competitive binding evaluation was undertaken with 2‐(4‐hydroxyphenylazo)benzoic acid (HABA) dye to provide visual verification of conjugation. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

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.     

Conjugation of quantum dots with graphene for fluorescence imaging of live cells

It is difficult to achieve fluorescent graphene-quantum dots (QDs) conjugation because graphene quenches the fluorescence of the QDs. In the present study, the conjugation of graphene (reduced graphene oxide, RGO) with QDs via a bridge of bovine serum albumin (BSA) provides a novel highly fluorescent nano probe for the first time. BSA capped QDs are firmly grafted onto polyethylenimine (PEI)/poly(sodium 4-styrenesulfonate) (PSS) coated RGO (graphene-QDs) via electrostatic layer by layer assembly. The strong luminescence of the graphene-QDs provides a potential for non-invasive optical in vitro imaging. The graphene-QDs are used for in vitro imaging of live human carcinoma (Hela) cells. Graphene-QDs could be readily up-taken by Hela cells in the absence of specific targeting molecules, e.g., antibodies or folic acid, and no in vitro cytotoxicity is observed at 360 μg mL(-1) of the graphene-QDs. The results for the imaging of live cells indicated that the cell-penetrating graphene-QDs could be a promising nano probe for intracellular imaging and therapeutic applications.