Sergey A. Dergunov

Permission to enter cell by shape: nanodisk vs nanosphere.

Changing polystyrene nanoparticles from three-dimensional spherical shape to two-dimensional disk shape promotes their cell surface binding with significant reduction of cell uptake. As a result of lower cell uptake, nanodisks show very little perturbations on cell functions such as cellular ROS generation, apoptosis and cell cycle progression compared to nanospheres. Therefore, disk-shaped nanoparticles may be a promising template for developing cell membrane-specific and safer imaging agents for a range of biomedical applications such as molecular imaging, tissue engineering, cell tracking, and stem cell separation.

Functionalization of Imprinted Nanopores in Nanometer-Thin Organic Materials†

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.

Nanocapsules with “invisible” walls

Nanometre-thin membranes, prepared by directed assembly within lipid bilayers, are capable of unhindered transport of ions while being impermeable to medium sized molecules.

pH-Mediated Catch and Release of Charged Molecules with Porous Hollow Nanocapsules

Here, we show that the charge of the nanopores in the nanometer-thin shells of hollow porous nanocapsules can regulate the transport of charged molecules. By changing the pH of external aqueous solution, we can entrap charged molecules in nanocapsules and trigger the release of encapsulated content.

Dye-loaded porous nanocapsules immobilized in a permeable polyvinyl alcohol matrix: a versatile optical sensor platform.

In this work we report on a versatile sensor platform based on encapsulated indicator dyes. Dyes are entrapped in hollow nanocapsules with nanometer-thin walls of controlled porosity. The porous nanocapsules retain molecules larger than the pore size but provide ultrafast access to their interior for molecules and ions smaller than the pore size. Dye-loaded nanocapsules are immobilized in a polyvinyl alcohol (PVA) matrix with high solvent permeability and rapid analyte diffusion. This approach provides robust sensing films with fast response and extended lifetime. To demonstrate the performance characteristics of such films, pH-sensitive indicator dyes were entrapped in vesicle-templated nanocapsules prepared by copolymerization of tert-butyl methacrylate, butyl methacrylate, and ethylene glycol dimethacrylate. As pH sensitive dyes, Nile blue A, bromophenol blue, and acid fuchsin were tested. Time-resolved absorbance measurements showed that the rate of the color change is controlled by the rate of diffusion of protons in the hydrogel. The pH-induced color change in a ~400 μm thick film is complete within 40 and 60 s. The porous nanocapsule loaded films showed excellent stability and reproducibility in long-term monitoring experiments. Compartmentalization of the indicator dyes within the nanocapsules increased their stability. The matrix caused a shift in the position of the color change of the dye compared to that in an aqueous buffer solution. The encapsulation/immobilization protocol described in this account is expected to be broadly applicable to a variety of indicator dyes in optical sensor applications.

Synergistic Co-Entrapment and Triggered Release in Hollow Nanocapsules with Uniform Nanopores

We describe a new co-entrapment and release motif based on the combination of noncovalent and steric interactions in materials with well-defined nanopores. Individual components enter hollow nanocapsules through nanopores in the capsule shell. Their complex, larger than the pore size, remains entrapped. The dissociation of the complex upon external stimulus releases entrapped components. Reversible formation of complexes between diaza-18-crown-6 and metal ions was used to demonstrate the feasibility of new approach to co-entrapment and triggered release.

Facile directed assembly of hollow polymer nanocapsules within spontaneously formed catanionic surfactant vesicles.

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.

Directed Assembly of Vesicle-Templated Polymer Nanocapsules under Near-Physiological Conditions

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.

Scattering studies of hydrophobic monomers in liposomal bilayers: an expanding shell model of monomer distribution.

Hydrophobic monomers partially phase separate from saturated lipids when loaded into lipid bilayers in amounts exceeding a 1:1 monomer/lipid molar ratio. This conclusion is based on the agreement between two independent methods of examining the structure of monomer-loaded bilayers. Complete phase separation of monomers from lipids would result in an increase in bilayer thickness and a slight increase in the diameter of liposomes. A homogeneous distribution of monomers within the bilayer would not change the bilayer thickness and would lead to an increase in the liposome diameter. The increase in bilayer thickness, measured by the combination of small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS), was approximately half of what was predicted for complete phase separation. The increase in liposome diameter, measured by dynamic light scattering (DLS), was intermediate between values predicted for a homogeneous distribution and complete phase separation. Combined SANS, SAXS, and DLS data suggest that at a 1.2 monomer/lipid ratio approximately half of the monomers are located in an interstitial layer sandwiched between lipid sheets. These results expand our understanding of using self-assembled bilayers as scaffolds for the directed covalent assembly of organic nanomaterials. In particular, the partial phase separation of monomers from lipids corroborates the successful creation of nanothin polymer materials with uniform imprinted nanopores. Pore-forming templates do not need to span the lipid bilayer to create a pore in the bilayer-templated films.

Blood triggered rapid release porous nanocapsules

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 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. ICG-loaded nanocapsules demonstrated 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.