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Co-localization of monomers, crosslinkers, and chain-transfer agents (CTA) within self-assembled bilayers in an aqueous suspension enabled the successful directed assembly of nanocapsules using a reversible addition-fragmentation chain transfer (RAFT) process without compromising the polymerization kinetics. This study uncovered substantial influence of the organized medium on the course of the reaction, including differential reactivity based on placement and mobility of monomers, crosslinkers, and CTAs within the bilayer.

An extensive body of work has documented the antioxidant role of xanthophylls (lutein and zeaxanthin) in human health and specifically how they provide photoprotection in human vision. More recently, evidence is emerging for the transcriptional regulation of antioxidant response by lutein/lutein cleavage products, similar to the role of β-carotene cleavage products in the modulation of retinoic acid receptors. Supplementation with xanthophylls also provides additional benefits for the prevention of age-related macular degeneration (AMD) and attenuation of Alzheimer’s disease symptoms. Mammalian β-carotene oxygenase 2 (BCO2) asymmetrically cleaves xanthophylls as well as β-carotene in vitro. We recently demonstrated that mouse BCO2 (mBCO2) is a functionally palmitoylated enzyme and that it loses palmitoylation when cells are treated with β-carotene. The mouse enzyme is the easiest model to study mammalian BCO2 because it has only one isoform, unlike human BCO2 with several major isoforms with various properties. Here, we used the same acyl-RAC methodology and confocal microscopy to elucidate palmitoylation and localization status of mBCO2 in the presence of xanthophylls. We created large unilamellar vesicle-based nanocarriers for the successful delivery of xanthophylls into cells. We demonstrate here that, upon treatment with low micromolar concentration of lutein (0.15 µM), mBCO2 is depalmitoylated and shows partial nuclear localization (38.00 ± 0.04%), while treatment with zeaxanthin (0.45 µM) and violaxanthin (0.6 µM) induces depalmitoylation and protein translocation from mitochondria to a lesser degree (20.00 ± 0.01% and 35.00 ± 0.02%, respectively). Such a difference in the behavior of mBCO2 toward various xanthophylls and its translocation into the nucleus in the presence of various xanthophylls suggests a possible mechanism for transport of lutein/lutein cleavage products to the nucleus to affect transcriptional regulation.

Vesicle-templated nanocapsules offer a unique combination of properties enabled by robust shells with single-nanometer thickness containing programmed uniform pores capable of fast and selective mass transfer. These capsules emerged as a versatile platform for creating functional devices, such as nanoreactors, nanosensors, and containers for the delivery of drugs and imaging agents. Nanocapsules are synthesized by a directed assembly method using self-assembled bilayers of vesicles as temporary scaffolds. In this approach, hydrophobic building blocks are loaded into the hydrophobic interior of vesicles formed from lipids or surfactants. Pore-forming templates are codissolved with the monomers and cross-linkers in the interior of the bilayer. The polymerization forms a cross-linked shell with embedded pore-forming templates. Removal of the surfactant scaffold and pore-forming templates leads to free-standing nanocapsules with shells containing uniform imprinted nanopores. Development of reliable and scalable synthetic methods for the modular construction of capsules with tunable properties has opened the opportunity to pursue practical applications of nanocapsules. In this Account, we discuss how unique properties of vesicle-templated nanocapsules translate into the creation of functional nanodevices. Specifically, we focus the conversation on applications aiming at the delivery of drugs and imaging agents, creation of fast-acting and selective nanoreactors, and fabrication of nanoprobes for sensing and imaging. We present a brief overview of the synthesis of nanocapsules with an emphasis on recent developments leading to robust synthetic methods including the synthesis under physiological conditions and creation of biodegradable nanocapsules. We then highlight unique properties of nanocapsules essential for practical applications, such as precise control of pore size and chemical environment, selective permeability, and ultrafast transport through the pores. We discuss new motifs for catch and release of small molecules with porous nanocapsules based on controlling the microenvironment inside the nanocapsules, regulating the charge on the orifice of nanopores in the shells, and reversible synergistic action of host and guest forming a supramolecular complex in nanocapsules. We demonstrate successful creation of fast-acting and selective nanoreactors by encapsulation of diverse homogeneous and nanoparticle catalysts. Due to unhindered flow of substrates and products through the nanopores, encapsulation did not compromise catalytic efficiency and, in fact, improved the stability of entrapped catalysts. We present robust nanoprobes based on nanocapsules with entrapped sensing agents and show how the encapsulation resulted in selective measurements with fast response times in challenging conditions, such as small volumes and complex mixtures. Throughout this Account, we highlight the advantages of encapsulation and discuss the opportunities for future design of nanodevices.

Vesicle-templated nanocapsules are prepared by polymerization of hydrophobic acrylic monomers and cross-linkers in the hydrophobic interior of self-assembled bilayers. Understanding the mechanism of capsule formation and the influence of synthetic parameters on the structural features and functional performance of nanocapsules is critical for the rational design of functional nanodevices, an emerging trend of application of the nanocapsule platform. This study investigated the relationship between basic parameters of the formulation and synthesis of nanocapsules and structural and functional characteristics of the resulting structures. Variations in the monomer/surfactant ratio, temperature of polymerization, and the molar fraction of the free-radical initiators were investigated with a multipronged approach, including shell thickness measurements using small-angle neutron scattering, evaluation of the structural integrity of nanocapsules with scanning electron microscopy, and determination of the retention of entrapped molecules using absorbance and fluorescence spectroscopy. Surprisingly, the thickness of the shells did not correlate with the monomer/surfactant ratio, supporting the hypothesis of substantial stabilization of the surfactant bilayer with loaded monomers. Decreasing the temperature of polymerization had no effect on the spherical structure of nanocapsules but resulted in progressively lower retention of entrapped molecules, suggesting that a spherical skeleton of nanocapsule forms rapidly, followed by filling the gaps to create the structure without pinholes. Lower content of initiators resulted in slower reactions, outlining the baseline conditions for practical synthetic protocols. Taken together, these findings provide insights into the formation of nanocapsules and offer methods for controlling the properties of nanocapsules in viable synthetic methods.

Nanocapsules with molecules threaded through the porous shells may lead to advanced cell-mimicking functional devices. Herein, we show the feasibility of synthesizing such hybrid nanostructures by using vesicle-templated polymer nanocapsules with controlled nanopores. Ship-in-a-bottle assembly inside a nanocapsule created an internal unit. An external unit was then connected to an entrapped internal unit through pre-attached linker threaded through a nanopore in the shell of the nanocapsule. Both internal and external units are larger than the pore size and cannot cross the shell, producing a rotaxane-like structure. Successful synthesis was achieved with fairly short linkers (six and ten carbon atoms in a chain), creating an opportunity for facile synthesis of functional devices capable of cross-shell communication.

Ground-state atomic oxygen [O(3P)] is an oxidant whose formation in solution was proposed but never proven. Polymer nanocapsules were used to physically separate dibenzothiophene S-oxide (DBTO), a source of O(3P), from an O(3P)-accepting molecule. Irradiation of polymer nanocapsules loaded with DBTO resulted in oxidation of the O(3P)-acceptor placed outside nanocapsules. The results rule out a direct oxygen atom transfer mechanism and are consistent with freely diffusing O(3P) as the oxidant.

Yolk-shell nanoreactors with metal nanoparticle core and ultrathin porous polymer shells are effective catalysts for heterogeneous reactions. Polymer shells provide size-selectivity and improved reusability of catalyst. Nanocapsules with single-nanometer porous shells are prepared by vesicle-templated directed assembly. Metal nanoparticles are formed either by selective initiation in pre-fabricated nanocapsules or simultaneously with the creation of a crosslinked polymer shell. In this study, we investigated the oxidation of benzyl alcohol and benzaldehyde catalyzed by gold nanoparticles and hydrogenation of cyclohexene catalyzed by platinum nanoparticles. Comparison of newly created nanoreactors with commercially available nanoparticles revealed superior reusability and size selectivity in nanoreactors while showing no negative effect on reaction kinetics.

Vesicle-templated nanocapsules have emerged as a viable platform for diverse applications. Shell thickness is a critical structural parameter of nanocapsules, where the shell plays a crucial role providing mechanical stability and control of permeability. Here we used small-angle neutron scattering (SANS) to determine the thickness of freestanding and surfactant-stabilized nanocapsules. Despite being at the edge of detectability, we were able to show the polymer shell thickness to be typically 1.0 ± 0.1 nm, which places vesicle-templated nanocapsules among the thinnest materials ever created. The extreme thinness of the shells has implications for several areas: mass-transport through nanopores is relatively unimpeded; pore-forming molecules are not limited to those spanning the entire bilayer; the internal volume of the capsules is maximized; and insight has been gained on how polymerization occurs in the confined geometry of a bilayer scaffold, being predominantly located at the phase-separated layer of monomers and cross-linkers between the surfactant leaflets.

Self-assembled gold monolayers offer several advantages for the realization of novel modified electrodes for biosensor applications. This is due to their ability to decrease non-specific adsorption and provide for covalent attachment of biomolecules. Surfaces for these applications require the precise control of ligand density, the ability to immobilize ligands, and in situ-modulation of ligand activity. In this study, we focused our studies on the immobilization of antibody on a gold monolayer surface. We self-assembled thioctic acid onto the gold surface as an anchor point for the immobilization of anti-fibrinogen onto the surface. The modifications to the gold surface were characterized by ELISA, ellipsometry, and AFM.

Yolk–shell nanostructures, or nanorattles, are created by growing metal nanoparticles exclusively inside hollow porous polymer nanocapsules. Metal ions enter the nanocapsules through size-selective nanopores. Synthesis of metal nanoparticles is initiated by an agent entrapped in nanocapsules. Tannic acid, β-cyclodextrin, and polyether dendrimer were used as sacrificial molecules for initiation and growth of gold nanoparticles. If needed, initiator molecules can be fragmented by acid hydrolysis and removed from nanocapsules. Variations in reaction conditions yield encapsulated nanoparticles with different size and shape. Further functionalization of nanorattles forms encapsulated core–shell nanoparticles.

Nanoprobes for surface-enhanced Raman scattering (SERS) were prepared by creating nanorattles, or yolk−shell structures, containing gold or silver nanoparticles entrapped in porous hollow polymer nanocapsules. Controlled permeability of the shells of nanocapsules, achieved by controlling the pore size and/or shell surface functionalization, resulted in size- and charge-selective SERS analyses. For example, a trace amount of phenanthroline, a model analyte, was detected in human blood plasma without preprocessing of plasma samples. Comparison with commercially available nanoparticles showed superior performance of the newly prepared nanorattle structures.

Gold nanoparticles entrapped in the hollow polymer nanocapsules undergo pH-mediated controlled aggregation. Encapsulated clusters of nanoparticles show absorbance at higher wavelengths compared with individual nanoparticles. The size of the aggregates is controlled by the number of nanoparticles entrapped in individual nanocapsules. Such controlled aggregation may permit small biocompatible nanoparticles exhibit desirable properties for biomedical applications that are typically characteristic of large nanoparticles.

We designed new terpolymers responsive to pH, temperature, and metal ions. Binding of metal ions amplifies the magnitude of thermal response. Variations in the pH and concentration of metal ions enable tuning the lower critical solution temperature (LCST) and thermosensitivity in a broad range. Upon changes in pH and/or temperature, polymers form associates with narrow size distribution ranging from less than 5 nm to more than 250 nm in diameter. The multistimuli-responsive properties of newly synthesized polymers are the result of combining hydrophobic, ionogenic, and non-charged hydrophilic moieties within the terpolymer structure. Polymers are produced by a potentially scalable method from inexpensive starting materials, 2-hydroxyethylacrylate (HEA), butylmethacrylate (BMA), and acrylic acid (AA). Polymers and their aggregates are characterized by NMR, dynamic light scattering, and electron microscopy. The ability to control the size of polymer aggregates and the magnitude of response to external stimuli opens exciting opportunities for practical applications.

Cross-linked polystyrene nanodisks were prepared by controlled polymerization of styrene and divinylbenzene in the interior of bicelles, discoidal lipid aggregates. Aggregation behavior of polymer nanodisks was studied in water, organic solvents, and solid phase. Nanodisks form stable dispersions in aqueous solutions of surfactants, such as sodium dodecyl sulfate (SDS). Varying SDS/nanodisk ratio allowed us to control the size of nanodisk aggregates. Nanodisks are readily solubilized in nonpolar organic solvents, such as toluene and carbon tetrachloride, to yield stable monodisperse suspensions. These findings open opportunities for creating nanodisk-based nanocomposite materials. Stable nanodisk suspension in toluene enabled small angle neutron scattering (SANS) measurements. SANS data confirmed the nanodisk diameter and allowed accurate measurement of nanodisk thickness (19.5 ± 1.0 A). In solid phase, nanodisks aggregate in sub-micron platelets.

Hollow polymer nanocapsules are produced by the polymerization within hydrophobic interior of lipid bilayers that act as temporary self-assembled scaffolds. Pore-forming templates are co-dissolved with monomers in the bilayers to create pores with controlled size and chemical environment. Polymerization was monitored with UV spectroscopy and dynamic light scattering. High resolution magic angle spinning NMR characterization provided detailed structural information about nanocapsules. Spherical shape was confirmed by electron microscopy. Medium-sized molecules can be entrapped within porous nanocapsules. No release of encapsulated molecules was observed within 240 days.

Robust surface-bound insulating polymer films with controlled thickness in

This work addresses the challenge of creating hollow polymer capsules with wall thickness in the single-nanometer range under mild conditions. We present a simple and scalable method for the synthesis of hollow polymer nanocapsules in the bilayers of spontaneously assembled surfactant vesicles. Polymerization is initiated thermally with the help of a peroxide initiator and an amine activator codissolved with monomers and cross-linkers in the hydrophobic interior of the surfactant bilayer. To avoid premature polymerization, the initiator and the activator were added separately to the mixtures of cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) containing monomers and cross-linkers. Upon hydration and mixing of the aqueous solutions, equilibrium monomer-loaded vesicles formed spontaneously after a brief incubation. The removal of oxygen and further incubation at slightly elevated temperatures (35-40 °C) for 1 to 2 h has led to the formation of hollow polymer nanocapsules. Structural and permeability characterization supported the high yield of nanocapsules with no pinhole defects.

Surfactant vesicles containing monomers in the interior of the bilayer were used to template hollow polymer nanocapsules. This study investigated the formation of surfactant/monomer assemblies by two loading methods, concurrent loading and diffusion loading. The assembly process and the resulting aggregates were investigated with dynamic light scattering, small angle neutron scattering, and small-angle X-ray scattering. Acrylic monomers formed vesicles with a mixture of cationic and anionic surfactants in a broad range of surfactant ratios. Regions with predominant formation of vesicles were broader for compositions containing acrylic monomers compared with blank surfactants. This observation supports the stabilization of the vesicular structure by acrylic monomers. Diffusion loading produced monomer-loaded vesicles unless vesicles were composed from surfactants at the ratios close to the boundary of a vesicular phase region on a phase diagram. Both concurrent-loaded and diffusion-loaded surfactant/monomer vesicles produced hollow polymer nanocapsules upon the polymerization of monomers in the bilayer followed by removal of surfactant scaffolds.

Surfactants and hydrophobic monomers spontaneously assemble into vesicles containing monomers within the bilayer. The joint action of monomers and surfactants is essential in this synergistic self-assembly. Polymerization in the bilayer formed hollow polymer nanocapsules.

Recently, we described the method for creating nanometer-thin organic materials with programmed size nanopores.[1] Controlling the pore geometry and mass transfer was identified as key to advances in DNA sequencing,[2] microreactors,[3] molecular electronics,[4] and drug-delivery devices.[5] Nanocapsules with selective permeability gained considerable attention in biomedical applications.[6] Controlling the chemical environment of nanopores is critical for realizing the full potential of nanothin porous materials.[7] Herein, we describe an efficient method for creating uniform nanopores with programmed chemical environment and demonstrate successful quantitative conversion of functional groups. Using lipid bilayers as temporary self-assembled scaffolds, we directed the assembly of sub-nanometer thin crosslinked organic polymer with embedded pore-forming templates (Figure 1). Previously, we used this method for creating nanocapsules with programmed size pores.[1] Modular construction of the template offers great versatility in varying the pore shape and size as well as the nature and number of functional groups. Figure 1 Directed assembly of nanothin polymer membranes with uniform imprinted nanopores. A) The self-assembled phospholipid bilayer is loaded with hydrophobic monomers and pore-forming templates. The template is a single molecule consisting of three parts: a ... Although liposomes[8] made of dimyristoylphosphatidylcholine (DMPC) were used in this work to demonstrate feasibility, we expect the method to be applicable to many other bilayers [9] The pore-forming template (1) was synthesized in one step from commercially available materials. Coupling of 1,2,3,4-tetra-O-acetyl-D-β-glucopyranose with 4-vinylbenzoic acid was performed by a standard protocol and produced the desired product in 85% yield (Scheme 1). Scheme 1 Synthesis of polymerizable and degradable pore-forming template (1). At lipid/template molar ratio of 34 or higher, all template molecules (1) are incorporated in the bilayer (Table 1). Following the loading of t-butylstyrene and divinylbenzene (1:1) and 1 into DMPC liposomes and UV-initiated polymerization, methanol was added to precipitate the nanocapsules and remove the lipids; nanocapsules were washed with methanol, resuspended in benzene, and freeze-dried. The FTIR spectrum of the capsules (Figure 2) revealed a characteristic peak at 1759 cm−1 corresponding to the C=O stretching of the ester groups. All template molecules (1) incorporated in the bilayer were found in the nanocapsules after the removal of lipids and multiple methanol washings (Table 1). FTIR spectra of nanocapsules revealed a shift of the band corresponding to the carbonyl group of 1 to higher wavenumbers (1759 cm−1 for 1 in nanocapsules vs. 1748 cm−1 for free 1, see Supporting Information), which is common for functional groups incorporated in bulk polymer.[10] These data agree with embedding 1 into the nanothin film as shown on Figure 1. Figure 2 FTIR spectra of the nanocapsules without pore-forming template (blue), nanocapsules with GPA after hydrolysis (purple), nanocapsules with 1 before (green) and after (red) hydrolysis. Table 1 Incorporation of 1 into the liposomal bilayer and nanocapsules. Alkaline hydrolysis of templates produced pores with free carboxylic groups (Figure 2). In control experiments, nanocapsules made without pore-forming templates and capsules made with glucose pentaacetate (GPA, a structural analog of 1 but without a polymerizable moiety) showed no signals corresponding to the carbonyl group after alkaline hydrolysis (Figure 2). Quantitative FTIR measurements (see Supporting Information) using aromatic C–H signal as an internal standard showed complete conversion of esters into carboxylic acids. We conclude that all templates 1 incorporated in the bilayer co-polymerized with the t-butylstyrene/divinylbenzene polymer matrix. We converted the carboxylic acid groups into the acid chloride by treatment with excess thionyl chloride and then formed two amides by the reaction of acid chloride with benzyl amine or 4-(aminomethyl)benzonitrile (Figure 3). We selected the latter to quantify the amount of amide using C≡N stretching vibration band at 2226 cm−1. The FTIR spectrum of the acid chloride shows the shift of the C=O band to 1774 cm−1 compared with 1763 cm−1 for the free acid (Figure 3) and absence of a band at 1215 cm−1 corresponding to the C(O)–O vibrations (see Supporting Information). The FTIR spectrum of the amide shows the C=O stretching vibrations at much lower frequency (1650 cm−1) than that of the free acid. Using the intensity of cyano group absorption band, we found that the carboxylic acid was completely converted to the amide. Figure 3 FTIR spectra of the nanocapsules with free carboxylic groups (blue), nanocapsules after reflux with thionyl chloride (red) and subsequent treatment with 4-(aminomethyl)-benzonitrile (green). If 34:1 ratio of DMPC to 1 is used, considering that area of each DMPC molecule is 62 A2,[11] the resulting nanoporous material has an estimated pore density of 9.5 × 1016 pores per m2 with an average distance between centers of pores of 3.2 nm and 3 × 103 pores per 100 nm capsule. Electron microscopy images demonstrated preservation of shape and integrity of nanocapsules (Figure 4). The size and shape of liposomes loaded with 1 and monomers (Figure 4a) are similar to those of liposomes containing polymerized capsules (Figure 4b). In agreement with previous reports,[1,8g] the detergent-assisted lipid removal did not affect the size distribution of nanocapsules (Figure 4d). Remarkably, SEM image shows clusters of nanocapsules with the same size after the lipid removal, hydrolysis, precipitation in methanol, multiple washings, resuspension in benzene, and freeze-drying (Figure 4c). These results suggest that the nanocapsules with nanometer-thin walls are stable under regular handling conditions such as solvent exchange. Figure 4 Electron micrographs of nanocapsules. a) TEM images of liposomes loaded with monomers, and b) polymer nanocapsules after polymerization. c) SEM image of polymer nanocapsules after hydrolysis and freeze-drying. d) TEM image of polymer nanocapsules after ... We used the previously described colored size probe retention assay to demonstrate the successful formation of nanopores with narrow size distribution.[1] We encapsulated a mixture of molecules with different colors and sizes in a liposome, carried out the polymerization, and separated the capsules from released probes on a size exclusion column. We used 0.6 nm yellow probe (methyl orange), 1.1 nm red probe (Procion Red), and 1.6 nm blue probe (1:1 β-cyclodextrin-Reactive Blue conjugate) to gauge the pore size.[1] All probes were retained in the capsules prior to template removal. After opening the pores, we observed complete release of 0.6 nm probes and retention of 1.1 nm and 1.6 nm probes (Figure 5) suggesting 0.8±0.2 nm pore size. Considering that size probe release from 100 nm capsules would occur faster than the chromatographic separation, quantitative retention of 1.1 nm and 1.6 nm probes allows us to conclude that very few capsules, if any, contain pinholes or pores larger than 1.1 nm. Figure 5 Selective permeability demonstrates successful formation of 0.8±0.2 nm pores. Nanocapsules were prepared with encapsulated colored size probe mixtures and separated on a size-exclusion column to remove released probes. The nanocapsule fraction ... The pore size is preserved even after freeze-drying the nanocapsules and resuspending them in water. When porous capsules containing 1.1 nm probes were dried, solubilized in 2% Triton X-100 solution and passed through a size-exclusion column, no release of the encapsulated size probes was observed. Combined with SEM images (Figure 4) this provides strong evidence that the materials preserve both their structure and function upon solvent exchange and drying. In summary, we demonstrated an efficient method for controlling the chemical environment of molecular-size pores in nanometer-thin organic materials. This method combines the use of temporary self-assembled scaffolds with molecular imprinting, which was widely used to fabricate functional materials.[12] We created uniformly sized pores with a single carboxylic functional group and quantitatively converted the carboxylic group into an acid chloride and subsequently into an amide. This opens opportunities for further functionalization for controlling the mass transfer across the pore, e.g. with stimuli-responsive moieties or creating arrays of functional groups that may potentially act as molecular recognition sites.

Rapid-release drug delivery systems present a new paradigm in emergency care treatments. Such systems combine a long shelf life with the ability to provide a significant dose of the drug to the bloodstream in the shortest period of time. Until now, development of delivery formulations has concentrated on slow release systems to ensure a steady concentration of the drug. To address the need for a quick release system, we created hollow polyacrylate nanocapsules with nanometer-thin porous walls. Burst release occurs upon interaction with blood components that leads to escape of the cargo. The likely mechanism of release involves a conformational change of the polymer shell caused by binding albumin. To demonstrate this concept, a near-infrared fluorescent dye indocyanine green (ICG) was incorporated inside the nanocapsules (NCs). ICG-loaded nanocapsules demonstrated a remarkable shelf life in aqueous buffers with no release of ICG for twelve months. Rapid release of the dye was demonstrated first in vitro using albumin solution and serum. SEM and light scattering analysis demonstrated the retention of the nanocapsule architecture after the release of the dye upon contact with albumin. In vivo studies using fluorescence lifetime imaging confirmed quick discharge of ICG from the nanocapsules following intravenous injection.

Size-selective pores in the shells of hollow polymer nanocapsules enable combined assembly and entrapment of molecules. Small building blocks enter the capsule through the pores. The assembled molecules, which are larger than the pores, remain entrapped in the nanocapsules. Porous nanometre-thin walls permit unhindered functionalization of entrapped molecules.