In this letter, we report a facile method to prepare robust phospholipid vesicles using commonly available phospholipids that are stabilized via formation of an interpenetrating, acid-labile, crosslinked polymer network, that imparts a site for controlled polymer destabilization and subsequent vesicle degradation. The polymer network was formed in the inner lamella of the phospholipid bilayer using 2-2-Di(methacryloyloxy-1-ethoxy)propane (DMOEP) and butyl methacrylate (BMA). Upon exposure to acidic conditions the highly crosslinked polymer network was partially converted to smaller linear polymers, resulting in substantially reduced vesicle stability upon exposure to chemical and physical insults. Isolated polymers showed a pH-dependent solubility in THF. Transmission electron microscopy, and dynamic light scattering showed time dependent enhanced vesicle stability in high concentrations of surfactant and vacuum conditions at elevated pH, whereas exposure to acidic pH rapidly decreased the vesicle stability, with complete destabilization observed in less than 24 hours. The resultant transiently stabilized vesicles may prove useful for enhanced drug delivery and chemical sensing applications and allow for improved physiological clearance.Phospholipid vesicles are routinely used for intracellular delivery, via fusion with the cell membrane and subsequent release of encapsulated cargo. While useful, vesicle delivery systems suffer from a series of physical limitations, including long-term stability and fusion with other substrates, resulting in inefficient cellular delivery of cargo. Transient stabilization of the phospholipid vesicle membrane may provide for better spatial and temporal control of cargo delivery and release. Whereas, vesicles prepared from naturally-occurring phospholipids offer limited stability in harsh chemical and biological environments, stabilization of the vesicle architecture allows many key advantages to be realized. 1-6 Vesicle stabilization has been achieved via several approaches, including: a) utilizing synthetic, polymerizable phospholipids, and b) polymer scaffolding -partitioning hydrophobic monomers into the vesicle lamella with subsequent polymerization. 1;4;6;7 The resulting stabilized vesicles can be loaded across cellular membranes intact, however, the irreversible stability severely limits cargo release, as well as physiological clearance. Chemically and mechanically robust vesicles that can be controllably destabilized under biologically relevant conditions are more desirable. In this letter, we report a facile method to prepare robust phospholipid vesicles using commonly available phospholipids that are stabilized via formation of an interpenetrating, acid-labile, cross-linked polymer network, that can be controllably destabilized and subsequently degraded.Stabilized 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles were prepared by fabricating an acid-cleavable, highly cross-linked copolymer within the hydrophobic lamella of the pre-organized vesicle assembly...

β-particle emitting radionuclides are useful molecular labels due to their abundance in biomolecules. Detection of β-emission from H,S, and P, important biological isotopes, is challenging due to the low energies (E ≤ 300 keV) and short penetration depths (≤0.6 mm) in aqueous media. The activity of biologically relevant β-emitters is usually measured in liquid scintillation cocktail (LSC), a mixture of energy-absorbing organic solvents, surfactants, and scintillant fluorophores, which places significant limitations on the ability to acquire time-resolved measurements directly in aqueous biological systems. As an alternative to LSC, we developed polystyrene-core, silica-shell nanoparticle scintillators (referred to as nanoSCINT) for quantification of low-energy β-particle emitting radionuclides directly in aqueous solutions. The polystyrene acts as an absorber for energy from emitted β-particles and can be loaded with a range of hydrophobic scintillant fluorophores, leading to photon emission at visible wavelengths. The silica shell serves as a hydrophilic shield for the polystyrene core, enabling dispersion in aqueous media and providing better compatibility with water-soluble analytes. While polymer and inorganic scintillating microparticles are commercially available, their large size and/or high density complicates effective dispersion throughout the sample volume. In this work, nanoSCINT nanoparticles were prepared and characterized. nanoSCINT responds to H,S, and P directly in aqueous solutions, does not exhibit a change in scintillation response between pH 3.0 and 9.5 or with 100 mM NaCl, and can be recovered and reused for activity measurements in bulk aqueous samples, demonstrating the potential for reduced production of LSC waste and reduced total waste volume during radionuclide quantification. The limits of detection for 1 mg/mL nanoSCINT are 130 nCi/mL forH, 8 nCi/mL for S, and<1 nCi/mL for P.

Nanomaterials have rapidly moved into the mainstream for chemical and biological analysis. Nanoparticle probes enhance signal intensity, increase the chemical and physical stability of the probe, and facilitate surface modification for specific targeting. Unfortunately, common problems are encountered with many nanoparticle probes, e.g., poor solubility, poor biocompatibility, and leakage of encapsulated components, that severely restrict the application of probes to ex vivo samples under carefully controlled conditions. A wide range of recently developed multifunctional nanomaterials are poised to make significant contributions to molecular analysis of biological systems. Composite nanoparticle geometries, including composites, hybrids, and core-shell nanoparticles prepared using two or more materials, e.g., silica/inorganic, silica/polymer, or polymer/inorganic combinations, offer improved solubility, easier functionalization, and decreased toxicity compared with the related single-component materials. Furthermore, composite nanomaterials present substantial signal amplification, and improved multiplexing for higher-sensitivity and higher-resolution measurements. Further development and integration of composite nanomaterials into the quantitative sciences will play a key role in the future of functional probes for imaging, quantitative analysis, and biological manipulation.

The inwardly rectifying K (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ± 3.3 pS), mean open times (τ = 0.72 ms, τ = 15.3 ms) and ATP affinity (IC = 115 ± 25 μM) when expressed in HEK293 cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-α-phosphatidylinositol-4,5-bisphosphate (PIP) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ± 0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ = 7.9 ms, τ = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC = 3.14 ± 0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.

β-Particle emitting radionuclides, such as 3H, 14C, 32P, 33P, and 35S, are important molecular labels due to their small size and the prevalence of these atoms in biomolecules but are challenging to selectively detect and quantify within aqueous biological samples and systems. Here, we present a core–shell nanoparticle-based scintillation proximity assay platform (nanoSPA) for the separation-free, selective detection of radiolabeled analytes. nanoSPA is prepared by incorporating scintillant fluorophores into polystyrene core particles and encapsulating the scintillant-doped cores within functionalized silica shells. The functionalized surface enables covalent attachment of specific binding moieties such as small molecules, proteins, or DNA that can be used for analyte-specific detection. nanoSPA was demonstrated for detection of 3H-labeled analytes, the most difficult biologically relevant β-emitter to measure due to the low energy β-particle emission, using three model assays that represent covalent and noncovalent binding systems that necessitate selectivity over competing 3H-labeled species. In each model, nmol quantities of target were detected directly in aqueous solution without separation from unbound 3H-labeled analyte. The nanoSPA platform facilitated measurement of 3H-labeled analytes directly in bulk aqueous samples without surfactants or other agents used to aid particle dispersal. Selectivity for bound 3H-analytes over unbound 3H analytes was enhanced up to 30-fold when the labeled species was covalently bound to nanoSPA, and 4- and 8-fold for two noncovalent binding assays using nanoSPA. The small size and enhanced selectivity of nanoSPA should enable new applications compared to the commonly used microSPA platform, including the potential for separation-free, analyte-specific cellular or intracellular detection.