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.