Silica coating of inorganic nanoparticles (NPs) is widely employed as a means of providing colloidal stability in aqueous media and surface functionality for a variety of applications, particularly ...in biology. When the NPs are synthesized with a surface coating of an organic surfactant like oleic acid, silica coating is performed by using the reverse microemulsion method. There are many reports in the literature of the successful application of this method to NaYF
upconversion NPs (doped with Yb and Er), and we have used this method to coat NaHoF
NPs designed as a mass cytometry reagent. This method failed when we attempted to apply it to other NaLnF
NPs (Ln = Sm, Eu, Tb). In this report we describe an investigation of the problem and show how it can be overcome. To control size in the synthesis of NaLnF
NPs and at the same time maintain size uniformity, it is necessary to adjust the Na/F and F/Ln ratios. Problems with silica coating are associated with substoichiometric F/Ln ratios (F/Ln < 4) that leave Ln oleate salts as a byproduct, often as a phase-separated oily layer that could not be purified from the NPs by precipitation with ethanol and redispersion in hexanes. The nature of the oily byproduct was inferred from a combination of TGA, NMR, and FTIR measurements. We explored five different additional purification procedures, and by adopting the appropriate purification method, NaLnF
NPs with a variety of compositions and synthesized using different reaction conditions could be coated with a thin shell of silica.
Micelles are formed when block copolymers are dissolved in solvents selective for one of the blocks. In contrast to micelles formed by surfactants, block copolymer micelles are generally more robust, ...and this opens the door to many applications. This article examines the formation and structure of fiber-like or filamentous micelles, with cross-sections of nanometer dimensions. These fascinating objects are currently under investigation for drug delivery applications, as impact modifiers for plastics, as templates for the deposition of metal nanoparticles and as precursors to nanoscale ceramics. Moreover, in some cases, studies of their formation and fragmentation are beginning to provide insight into the generation of protein fibers, such as actin or amyloid fibers, derived from soluble cytosolic protein precursors.
Mass cytometry (MC) is a bioanalytical technique that uses metal-tagged antibodies (Abs) for high-dimensional single-cell immunoassays. Currently, this technology can measure over 40 parameters ...simultaneously on individual cells using metal-chelating polymer (MCP) based reagents. However, MC can in principle detect up to 135 parameters with the development of new elemental mass tags. Here we report the development of a tantalum oxide nanoparticle (NP)-based mass tag for MC immunoassays. Uniform-sized amine-functionalized tantalum oxide NPs (d ∼ 5.7 nm) were synthesized via a one-pot two-step reverse microemulsion method. These amine-functionalized NPs were further modified with azide groups by reacting with azide-PEG2k succinimidyl carboxymethyl ester (NHS-PEG2k -N3) cross-linkers. The Ab-NP conjugates were prepared by reacting azide-functionalized NPs with dibenzocyclooctyne (DBCO)-functionalized primary or secondary Abs (DBCO-Ab) followed by fast protein size exclusion liquid chromatography (FPLC) purification. Three Ab-NP conjugates (TaO2-PEG2k -goat antimouse, TaO2-PEG2k -CD25, TaO2-PEG2k -CD196) were fabricated and tested in MC immunoassays. For the TaO2-PEG2k -goat antimouse conjugate, we showed that it can effectively detect abundant CD20 biomarkers on Ramos cells. For TaO2-PEG2k -CD25 and TaO2-PEG2k -CD196 conjugates, we demonstrated that these Ab-NP conjugates could be integrated into the commercial Ab staining panels for high-dimensional single-cell immune profiling of human peripheral blood mononuclear cells.
Monodisperse fiber-like micelles with a crystalline π-conjugated polythiophene core with lengths up to ca. 700 nm were successfully prepared from the diblock copolymer ...poly(3-hexylthiophene)-block-polystyrene using a one-dimensional self-seeding technique. Addition of a polythiophene block copolymer with a different corona-forming block to the resulting nanofibers led to the formation of segmented B-A-B triblock co-micelles by crystallization-driven seeded growth. The key to these advances appears to be the formation of a relatively defect-free crystalline micelle core under the self-seeding conditions.
Non-spherical nanostructures derived from soft matter and with uniform size-that is, monodisperse materials-are of particular utility and interest, but are very rare outside the biological domain. We ...report the controlled formation of highly monodisperse cylindrical block copolymer micelles (length dispersity < or = 1.03; length range, approximately 200 nm to 2 microm) by the use of very small (approximately 20 nm) uniform crystallite seeds that serve as initiators for the crystallization-driven living self-assembly of added block-copolymer unimers with a crystallizable, core-forming metalloblock. This process is analogous to the use of small initiator molecules in classical living polymerization reactions. The length of the nanocylinders could be precisely controlled by variation of the unimer-to-crystallite seed ratio. Samples of the highly monodisperse nanocylinders of different lengths that are accessible using this approach have been shown to exhibit distinct liquid-crystalline alignment behaviour.
We present a set of model-independent differential equations to analyze isothermal titration calorimetry (ITC) experiments. In contrast with previous approaches that begin with specific assumptions ...about the number of binding sites and the interactions among them (e.g., sequential, independent, cooperative), our derivation makes more general assumptions, such that a receptor with multiple sites for one type of ligand species (homotropic binding) can be studied with the same analytical expression. Our approach is based on the binding polynomial formalism, and the resulting analytical expressions can be extended to account for any number of binding sites and any type of binding interaction among them. We refer to the set of model-independent differential equations to study ITC experiments as a differential binding model (DBM). To demonstrate the flexibility of our DBM, we present the analytical expressions to study receptors with one or two binding sites. The DBM for a receptor with one site is equivalent to the Wiseman isotherm but with a more intuitive representation that depends on the binding polynomial and the dimensionless parameter c = K·M T, where K is the binding constant and M T the total receptor concentration. In addition, we show how to constrain the general DBM for a receptor with two sites to represent sequential, independent, or cooperative binding interactions between the sites. We use the sequential binding model to study the binding interaction between Gd(III) and citrate anions. In addition, we simulate calorimetry titrations of receptors with positive, negative, and noncooperative interactions between the two binding sites. Finally, we derive a DBM for titrations of receptors with n-independent binding sites.
The efficient penetration of drug nanocarriers into tumors is an important prerequisite for therapeutic and diagnostic success. The physicochemical properties of nanocarriers, including size, shape ...and surface chemistry have been shown to influence their transport in biological systems. Recent studies have shown that elongated nanoparticles (NPs) can exhibit advantageous properties in comparison to spherical NPs, but these experiments have involved a variety of different materials, many of which are characterized by a broad size distribution. Here we describe a series of rigid rod-like micelles of uniform width, with narrow length distributions, and common surface chemistry, and examine their cell uptake and penetration into multicellular tumor spheroids (MCTSs) formed from two human breast cancer cell lines (MDA-MB-436 and MDB-MB-231). These micelles were prepared from a polyferrocenylsilane (PFS) diblock copolymer (BCP) with a corona block consisting of a statistical polymer of aminopropyl methacrylamide and oligo(ethyleneglycol methacrylate) (PFS
-b-PAPMA
-stat-POEGMA
). The rigid rod micelles, with a common width (12 nm) and lengths ranging from 80 to 2000 nm, were prepared by seeded growth crystallization-driven self-assembly in ethanol and then transferred to water. To consider whether changing the shape of these micelles affects its uptake and penetration behavior, analogous spherical micelles were prepared by direct nanoprecipitation into water. Both micelle shape and length were found to influence cellular uptake and penetration into 500 μm MCTSs. Laser confocal fluorescence microscopy was used to examine penetration of these micelles into three-dimensional MCTS up to 90 μm depth. Micelles with an elongated shape and short length (80 nm) demonstrated the deepest penetration into the MCTSs formed by MDA-MB-436 cells. Micelles with lengths of 200 nm also showed substantial penetration into these MCTS, but the extent and depth of tumor penetration of the rod-like micelles decreased with increasing aspect ratio. MCTS of MDA-MB-231 cells had a less dense, more open structure than those formed by MDA-MB-436 cells. Here more extensive penetration was observed, particularly for the longer micelle samples.
Mass cytometry (MC) is a high throughput multiparameter analytical technique for determining biomarker expression in cells. In MC, antibodies (Abs) are tagged with heavy metal isotopes
conjugation to ...metal chelating polymers (MCPs). To improve the sensitivity of MC towards low abundance biomarkers, we are developing nanoparticle (NP)-based reagents as mass tags for Abs. We examine the use of silica-coated NaHoF
NPs (
∼ 12 nm) decorated with PEG5k conjugated to thiol-modified primary or secondary Abs for MC assays. We compare the sensitivity of NP-Ab conjugates to MCP-Ab conjugates towards seven biomarkers with varying expression levels across six cell lines. We also perform a multi-parameter assay using a cocktail of both NP- and MCP-based reagents to detect seven cellular markers in peripheral blood mononuclear cells (PBMCs). In the case of highly abundant markers, signal enhancements from NP-Ab conjugates offer minimal advantages over MCP-Ab conjugates, which already give strong signals. In the case of biomarkers with lower abundance, the level of signal enhancements depended on the nature of the biomarker being detected, or on the type of detection method used. When comparing the indirect detection of CD14 on THP-1 cells using NPs or MCPs conjugated to secondary Abs, the NP reagents offered little signal enhancements compared to the MCP reagents. However, in the case of direct CD14 detection on THP-1 or U937 cells using NPs or MCPs conjugated to primary Abs, a 30- or 450-fold signal enhancement was seen from the NP-based reagent. In the experiments where both NP-Ab and MCP-Ab conjugates were used together to stain PBMCs, we found that the presence of the NP-Ab conjugates did not affect the function of MCP-Ab conjugates, and the NP-Ab conjugates showed minimal non-specific interaction with cells without the target biomarker (CD14). Furthermore, these NP-Ab conjugates could be used to identify rare CD14
monocytes from the PBMC mixture with a 20-fold signal increase when compared to the use of only MCP-Ab conjugates. Collectively, the strong signal amplification obtained from NP reagents demonstrate the potential of these reagents to be used in conjunction with MCP-reagents to detect rare cellular markers or cell types that may otherwise be overlooked when using MCP-reagents alone.
Bottom-up fabrication protocols for uniform 3D hierarchical structures in solution are rare. We report two different approaches to fabricate uniform 3D spherulites and their precursors using mixtures ...of poly(ferrocenyldimethylsilane) (PFS) block copolymer (BCP) and PFS homopolymer (HP). Both protocols are designed to promote defects in 2D assemblies that serve as intermediate structures. In a multistep seeded growth protocol, we add the BCP/HP mixture to (1D) rod-like PFS micelles in a selective solvent as first-generation seeds. This leads to 2D platelet structures. If this step is conducted at a high supersaturation, secondary crystals form on the basal surface of these platelets. Co-crystallization and rapid crystallization of BCP/HP promote the formation of defects that act as nucleation sites for secondary crystals, resulting in multilayer platelets. This is the key step. The multilayer platelets serve as second-generation seeds upon subsequent addition of BCP/HP blends and, with increasing supersaturation, lead to the sequential formation of uniform (3D) hedrites, sheaves, and spherulites. Similar structures can also be obtained by a simple one-pot direct self-assembly (heating–cooling–aging) protocol of PFS BCP/HP blends. In this case, for a carefully chosen but narrow temperature range, PFS HPs nucleate formation of uniform structures, and the annealing temperature regulates the supersaturation level. In both protocols, the competitive crystallization kinetics of HP/BCP affects the morphology. Both protocols exhibit broad generality. We believe the morphological transformation from 2D to 3D structures, regulated by defect formation, co-crystallization, and supersaturation levels, could apply to various semicrystalline polymers. Moreover, the 3D structures are sufficiently robust to serve as recoverable carriers for nanoparticle catalysts, exhibiting valuable catalytic activity and opening new possibilities for applications requiring exquisite 3D structures.
Living crystallization-driven self-assembly in solution has proven to be an excellent method to prepare core-crystalline micelles with an exquisite control over their size and morphology. While ...numerous studies have been performed to test their assembly in various media for potential applications, their stability under different stimuli remains yet to be established. In the present study, we performed light scattering and transmission electron microscopy experiments to investigate the effect of concentration on the self-seeding of core-crystalline one-dimensional (1D) seed crystallites with a long corona forming block. As previously reported, the seed crystallites dissolve much easier at low concentrations than at relatively high concentrations. We also observe that for all concentrations, the micelle width broadens during annealing. To explain these results, we developed a model based on a scaling approach. We show that the growth of ribbon-like core-crystalline micelles depends on the distance between the corona chains grafted on the micelle core, the thickness of the core, and, more surprisingly, on the number of chains constituting the core. This scaling approach also allowed us to explain how the interactions between the corona-forming block and the solvent influence the behavior of the seed crystallites at different annealing temperatures and concentrations. This model should thus prove very useful to understand and predict the effect of different media and stimuli on the size, morphology, and stability of core-crystalline micelles.
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