Design, synthesis, and spectroscopic profiling of novel coumarin dyes: Investigating solvent sensitivity and photophysical properties.

Coumarin dyes are highly versatile and widely employed as fluorescent chemosensors in a variety of fields, including molecular imaging, bioorganic chemistry, analytical chemistry, materials chemistry, biology, and medical science. Thanks to their outstanding photostability and high quantum yield, they represent an ideal choice for developing sensitive and selective sensing platforms. In this study, we successfully designed and synthesized four new dyes based on the coumarin dye molecular skeleton, investigating their solvent sensitivity and spectroscopic properties. Our novel coumarin dyes were synthesized by a straightforward approach, reacting coumarin-3-carboxylic acid succinimidyl ester derivatives with corresponding amines in 1,4-dioxane as a solvent. We carefully monitored the completion of the reactions using thin-layer chromatography (TLC) and characterized these dyes using spectral and elemental analyses. We further investigated the UV, fluorescence, time-correlated single photon counting (TCSPC) technique and time-resolved spectroscopy (TRES) of these dyes in different solvents and on polymer film poly(methyl methacrylate) (PMMA). The quantum yield of the synthesized dyes was determined, with values observed to range between 0.55 and 0.94. Most of the dye-solvent and dye-polymer combinations exhibited single exponential decay, with lifetimes ranging from 2.3 to 3 ns. Minor deviations from single exponential behavior were observed for most of the dyes in toluene, while significant deviations were observed for coumarin dyes with piperazine moiety. We have provided a rationalization of these results in terms of the chemical functionalities of the various dyes. Furthermore, we investigated the effect of interactions between 7-methoxy-2-oxo-N-(2-(piperazin-1-yl)ethyl)-2H-chromene-3-carboxamide and silica nanoparticles (Ludox) on the spectroscopic properties of these dyes, with charge transfer being one possible mechanism contributing to the behavior of the dyes. Additionally, we explored the effect of trifluoroacetic acid (TFA) on the dyes' emission intensity and fluorescence decay. Based on our UV and fluorescence measurements of the dyes in different solvents, we have concluded that these dyes can create excellent donor-acceptor pairs for our upcoming fluorescence resonance energy transfer (FRET) experiments.

Design Parameters for a Mass Cytometry Detectable HaloTag Ligand.

Mass cytometry permits the high dimensional analysis of complex biological samples; however, some techniques are not yet integrated into the mass cytometry workflow due to reagent availability. The use of self-labeling protein systems, such as HaloTag, are one such application. Here, we describe the design and implementation of the first mass cytometry ligands for use with HaloTag. "Click"-amenable HaloTag warheads were first conjugated onto poly(l-lysine) or poly(acrylic acid) polymers that were then functionalized with diethylenetriaminepentaacetic acid (DTPA) lutetium metal chelates. Kinetic analysis of the HaloTag labeling rates demonstrated that the structure appended to the 1-chlorohexyl warhead was key to success. A construct with a diethylene glycol spacer appended to a benzamide gave similar rates (kobs ∼ 102 M-1 s-1), regardless of the nature of the polymer. Comparison of the polymer with a small molecule chelate having rapid HaloTag labeling kinetics (kobs ∼ 104 M-1 s-1) suggests the polymers significantly reduced the HaloTag labeling rate. HEK293T cells expressing surface-exposed GFP-HaloTag fusions were labeled with the polymeric constructs and 175Lu content measured by cytometry by time-of-flight (CyTOF). Robust labeling was observed; however, significant nonspecific binding of the constructs to cells was also present. Heavily pegylated polymers demonstrated that nonspecific binding could be reduced to allow cells bearing the HaloTag protein to be distinguished from nonexpressing cells.

Defect-Induced Secondary Crystals Drive Two-Dimensional to Three-Dimensional Morphological Evolution in the Co-Self-Assembly of Polyferrocenylsilane Block Copolymer and Homopolymer.

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.

Hypersensitive MR angiography based on interlocking stratagem for diagnosis of cardiac-cerebral vascular diseases.

Magnetic resonance (MR) angiography is one of the main diagnostic approaches for cardiac-cerebral vascular diseases. Nevertheless, the non-contrast-enhanced MR angiography suffers from its intrinsic problems derived from the blood flow-dependency, while the clinical Gd-chelating contrast agents are limited by their rapid vascular extravasation. Herein, we report a hypersensitive MR angiography strategy based on interlocking stratagem of zwitterionic Gd-chelate contrast agents (PAA-Gd). The longitudinal molar relaxivity of PAA-Gd was 4.6-times higher than that of individual Gd-chelates as well as appropriate blood half-life (73.8 min) and low immunogenicity, enabling sophisticated micro-vessels angiography with a resolution at the order of hundred micrometers. A series of animal models of cardiac-cerebrovascular diseases have been built for imaging studies on a 7.0 T MRI scanner, while the clinical translation potential of PAA-Gd has been evaluated on swine on a 3.0 T clinical MRI scanner. The current studies offer a promising strategy for precise diagnosis of vascular diseases.

Polyferrocenylsilane Block Copolymer Spherulites in Dilute Solution.

Self-assembly of block copolymers (BCP) into uniform 3D structures in solution is an extremely rare phenomenon. Furthermore, the investigation of general prerequisites for fabricating a specific uniform 3D structure remains unknown and challenging. Here, through a simple one-pot direct self-assembly (heating and cooling) protocol, we show that uniform spherulite-like structures and their precursors can be prepared with various poly(ferrocenyldimethylsilane) (PFS) BCPs in a variety of polar and non-polar solvents. These structures all evolve from elongated lamellae into hedrites, sheaf-like micelles, and finally spherulites as the annealing temperature and supersaturation degree are increased. The key feature leading to this growth trajectory is the formation of secondary crystals by self-nucleation on the surface of early-elongated lamellae. We identified general prerequisites for fabricating PFS BCP spherulites in solution. These include corona/PFS core block ratios in the range of 1-5.5 that favor the formation of 2D structures as well as the development of secondary crystals on the basal faces of platelets at early stages of the self-assembly. The one-pot direct self-assembly provides a general protocol to form uniform spherulites and their precursors consisting of PFS BCPs that match these prerequisites. In addition, we show that manipulation of various steps in the direct self-assembly protocol can regulate the size and shape of the structures formed. These general concepts show promise for the fabrication and optimization of spherulites and their precursors from semicrystalline BCPs with interesting optical, electronic, or biomedical properties using the one-pot direct self-assembly protocol.

Reagents for Mass Cytometry.

Mass cytometry (cytometry by time-of-flight detection [CyTOF]) is a bioanalytical technique that enables the identification and quantification of diverse features of cellular systems with single-cell resolution. In suspension mass cytometry, cells are stained with stable heavy-atom isotope-tagged reagents, and then the cells are nebulized into an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) instrument. In imaging mass cytometry, a pulsed laser is used to ablate ca. 1 µm2 spots of a tissue section. The plume is then transferred to the CyTOF, generating an image of biomarker expression. Similar measurements are possible with multiplexed ion bean imaging (MIBI). The unit mass resolution of the ICP-TOF-MS detector allows for multiparametric analysis of (in principle) up to 130 different parameters. Currently available reagents, however, allow simultaneous measurement of up to 50 biomarkers. As new reagents are developed, the scope of information that can be obtained by mass cytometry continues to increase, particularly due to the development of new small molecule reagents which enable monitoring of active biochemistry at the cellular level. This review summarizes the history and current state of mass cytometry reagent development and elaborates on areas where there is a need for new reagents. Additionally, this review provides guidelines on how new reagents should be tested and how the data should be presented to make them most meaningful to the mass cytometry user community.

Treatment of Orthotopic U251 Human Glioblastoma Multiforme Tumors in NRG Mice by Convection-Enhanced Delivery of Gold Nanoparticles Labeled with the ß-Particle-Emitting Radionuclide, 177Lu.

In this study, we investigated convection-enhanced delivery (CED) of 23 ± 3 nm gold nanoparticles (AuNPs) labeled with the ß-particle-emitting radionuclide 177Lu (177Lu-AuNPs) for treatment of orthotopic U251-Luc human glioblastoma multiforme (GBM) tumors in NRG mice. The cytotoxicity in vitro of 177Lu-AuNPs (0.0-2.0 MBq, 4 × 1011 AuNPs) on U251-Luc cells was also studied by a clonogenic survival assay, and DNA double-strand breaks (DSBs) caused by ß-particle emissions of 177Lu were measured by confocal immunofluorescence microscopy for γH2AX. NRG mice with U251-Luc tumors in the right cerebral hemisphere of the brain were treated by CED of 1.1 ± 0.2 MBq of 177Lu-AuNPs (4 × 1011 AuNPs). Control mice received unlabeled AuNPs or normal saline. Tumor retention of 177Lu-AuNPs was assessed by single-photon emission computed tomography/computed tomography (SPECT/CT) imaging and biodistribution studies. Radiation doses were estimated for the tumor, brain, and other organs. The effectiveness for treating GBM tumors was determined by bioluminescence imaging (BLI) and T2-weighted magnetic resonance imaging (MRI) and by Kaplan-Meier median survival. Normal tissue toxicity was assessed by monitoring body weight and hematology and blood biochemistry analyses at 14 d post-treatment. 177Lu-AuNPs (2.0 MBq, 4 × 1011 AuNPs) decreased the clonogenic survival of U251-Luc cells to 0.005 ± 0.002 and increased DNA DSBs by 14.3-fold compared to cells treated with unlabeled AuNPs or normal saline. A high proportion of 177Lu-AuNPs was retained in the U251-Luc tumor in NRG mice up to 21 d with minimal re-distribution to the brain or other organs. The radiation dose in the tumor was high (599 Gy). The dose in the normal right cerebral hemisphere of the brain excluding the tumor was 93-fold lower (6.4 Gy), and 2000-3000-fold lower doses were calculated for the contralateral left cerebral hemisphere (0.3 Gy) or cerebellum (0.2 Gy). The doses in peripheral organs were <0.1 Gy. BLI revealed almost complete tumor growth arrest in mice treated with 177Lu-AuNPs, while tumors grew rapidly in control mice. MRI at 28 d post-treatment and histological staining showed no visible tumor in mice treated with 177Lu-AuNPs but large GBM tumors in control mice. All control mice reached a humane endpoint requiring sacrifice within 39 d (normal saline) or 45 d post-treatment (unlabeled AuNPs), while 5/8 mice treated with 177Lu-AuNPs survived up to 150 d. No normal tissue toxicity was observed in mice treated with 177Lu-AuNPs. We conclude that CED of 177Lu-AuNPs was highly effective for treating U251-Luc human GBM tumors in the brain in NRG mice at amounts that were non-toxic to normal tissues. These 177Lu-AuNPs administered by CED hold promise for treating patients with GBM to prevent recurrence and improve long-term outcome.

Development of Multiplexed Bead-Based Immunoassays for Profiling Soluble Cytokines and CD163 Using Mass Cytometry.

Bead-based immunoassays are multiparametric analysis allowing for the simultaneous quantification of a large number of biomarkers within a single sample. Mass cytometry is an emerging cytometric technique that offers a high multiplexing capacity in a high-throughput setting but has not yet been applied to bead-based assays. In this study, we developed a multiplex bead-based immunoassay of cytokines and CD163 designed for mass cytometry (MC). A set of 11 types of lanthanide-encoded microbeads were synthesized by two-stage dispersion polymerization as classifier candidates for the assay. These beads were then decorated with different Abs on the surface to capture the target cytokines in solution. Gold nanoparticles were employed as reporters to identify the binding of target cytokines on the classifier surface. As a proof-of-concept study, we first developed four-plex and nine-plex assays of mixtures of cytokines in standard solutions. The MC signal intensities of these immunoassays were responsive to the concentration differences in the standard solutions with high detection sensitivities at low analyte concentrations. Finally, we examined a sample of peripheral blood mononuclear cells (PBMCs) with the nine-plex assay, comparing an unstimulated sample with a sample stimulated to promote cytokine secretion.

90Y-Labeled Gold Nanoparticle Depot (NPD) Combined with Anti-PD-L1 Antibodies Strongly Inhibits the Growth of 4T1 Tumors in Immunocompetent Mice and Induces an Abscopal Effect on a Distant Non-Irradiated Tumor.

The effectiveness and normal tissue toxicity of a novel nanoparticle depot (NPD) brachytherapy seed incorporating gold nanoparticles (AuNPs) labeled with ß-particle emitting, 90Y (termed a "radiation nanomedicine"), were studied for the treatment of 4T1 triple-negative murine mammary carcinoma tumors in Balb/c mice and for inducing an abscopal effect on a distant non-irradiated tumor alone or combined with anti-PD-L1 immune checkpoint antibodies. Balb/c mice with two subcutaneous 4T1 tumors─a primary tumor and a distant secondary tumor were implanted intratumorally (i.t.) in the primary tumor with NPD incorporating 3.5 MBq of 90Y-AuNPs (1 × 1014 AuNPs) or unlabeled AuNPs, alone or combined with systemically administered anti-PD-L1 antibodies (200 µg i.p. three times/week for 2 weeks) or received anti-PD-L1 antibodies alone or no treatment. The primary tumor was strongly growth-inhibited over 14 d by NPD incorporating 90Y-AuNPs but only very modestly inhibited by NPD incorporating unlabeled AuNPs. Anti-PD-L1 antibodies alone were ineffective, and combining anti-PD-L1 antibodies with NPD incorporating 90Y-AuNPs did not further inhibit the growth of the primary tumor. Secondary tumor growth was inhibited by treatment of the primary tumor with NPD incorporating 90Y-AuNPs, and growth inhibition was enhanced by anti-PD-L1 antibodies. Treatment of the primary tumor with NPD incorporating unlabeled AuNPs or anti-PD-L1 antibodies alone had no effect on secondary tumor growth. Biodistribution studies showed high uptake of 90Y in the primary tumor [516-810% implanted dose/g (%ID/g)] but very low uptake in the secondary tumor (0.033-0.16% ID/g) and in normal tissues (<0.5% ID/g) except for kidneys (5-8% ID/g). Very high radiation absorbed doses were estimated for the primary tumor (472 Gy) but very low doses in the secondary tumor (0.13 Gy). There was highdose-heterogeneity in the primary tumor with doses as high as 9964 Gy in close proximity to the NPD, decreasing rapidly with distance from the NPD. Normal organ doses were low (<1 Gy) except for kidneys (4 Gy). No normal tissue toxicity was observed, but white blood cell counts (WBC) decreased in tumor-bearing mice treated with NPD incorporating 90Y-AuNPs. Decreased WBC counts were interpreted as tumor response and not toxicity since these were higher than that in healthy non-tumor-bearing mice, and there was a direct association between WBC counts and 4T1 tumor burden. We conclude that implantation of NPD incorporating 90Y-AuNPs into a primary 4T1 tumor in Balb/c mice strongly inhibited tumor growth and combined with anti-PD-L1 antibodies induced an abscopal effect on a distant secondary tumor. This radiation nanomedicine is promising for the local treatment of triple-negative breast cancer tumors in patients, and these therapeutic effects may extend to non-irradiated lesions, especially when combined with checkpoint immunotherapy.

Changing Surface Polyethylene Glycol Architecture Affects Elongated Nanoparticle Penetration into Multicellular Tumor Spheroids.

Nanoparticles (NPs) designed for biomedical applications are coated with protein-repellent polymers. Here, we examine the penetration of rodlike NPs with narrow size distributions (Ln = 170 nm, wn = 12 nm) into multicellular tumor spheroids prepared from two human cancer cell lines. Two types of NPs with different core materials [polyferrocenylsilane and cellulose nanocrystals (CNC)] were coated with a dense brush of poly(oligoethyleneglycol methacrylate) (POEGMA), while a second CNC NP sample was coated with a linear polyethylene glycol (PEG) brush. While the core material had little influence, the coating material was strikingly important, with POEGMA-coated NPs penetrating much more deeply into the tumor spheroids than the NPs coated with linear PEG. Localization experiments using 111In-labeled POEGMA-coated CNC NPs showed that most of the radioactivity remained in the interstitial space (ca. 78%) with little cell uptake (ca. 6%). Hence, the deep penetration of these nanorods into tumor spheroids is associated with an interstitial diffusion pathway through the extracellular matrix and not cellular transcytosis.

Polymeric dipicolylamine based mass tags for mass cytometry.

Mass cytometry is an emerging powerful bioanalytical technique for high-dimensional single-cell analysis. In this technique, cells are stained with metal-isotope-tagged antibodies and are analyzed by an inductively coupled plasma time-of-flight mass spectrometer. While there are more than 100 stable isotopes available in the m/z 75 to 209 detection range of the instrument, only about 50 parameters can be measured per cell because current reagents are metal-chelating polymers with pendant aminocarboxylate chelators that only bind hard metal ions such as the rare earths and Bi3+. Here we describe the synthesis and characterization of a new type of metal-chelating polymer with pendant dipicolylamine chelators suited to binding intermediate to soft metals such as rhenium and platinum. We introduce two different conjugation strategies, a thiol-maleimide reaction that works well for rhenium, and a DBCO-azide click reaction designed to avoid potential complications of Pt and other heavy metals interacting with thiol groups. We show that these polymers can serve as new elemental mass tags for mass cytometry. Antibody-polymer conjugates of CD20 and CD8a prepared by both coupling reactions were employed in conjunction with commercial metal-conjugated antibodies for multi-parameter single-cell immunoassays.

Effect of Excess Ligand on the Reverse Microemulsion Silica Coating of NaLnF4 Nanoparticles.

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 NaYF4 upconversion NPs (doped with Yb and Er), and we have used this method to coat NaHoF4 NPs designed as a mass cytometry reagent. This method failed when we attempted to apply it to other NaLnF4 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 NaLnF4 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, NaLnF4 NPs with a variety of compositions and synthesized using different reaction conditions could be coated with a thin shell of silica.

The role of cooling rate in crystallization-driven block copolymer self-assembly.

Self-assembly of crystalline-coil block copolymers (BCPs) in selective solvents is often carried out by heating the mixture until the sample appears to dissolve and then allowing the solution to cool back to room temperature. In self-seeding experiments, some crystallites persist during sample annealing and nucleate the growth of core-crystalline micelles upon cooling. There is evidence in the literature that the nature of the self-assembled structures formed is independent of the annealing time at a particular temperature. There are, however, no systematic studies of how the rate of cooling affects self-assembly. We examine three systems based upon poly(ferrocenyldimethylsilane) BCPs that generated uniform micelles under typical conditions where cooling took pace on the 1-2 h time scale. For example, several of the systems generated elongated 1D micelles of uniform length under these slow cooling conditions. When subjected to rapid cooling (on the time scale of a few minutes or faster), branched structures were obtained. Variation of the cooling rate led to a variation in the size and degree of branching of some of the structures examined. These changes can be explained in terms of the high degree of supersaturation that occurs when unimer solutions at high temperature are suddenly cooled. Enhanced nucleation, seed aggregation, and selective growth of the species of lowest solubility contribute to branching. Cooling rate becomes another tool for manipulating crystallization-driven self-assembly and controlling micelle morphologies.

Biotinylated Lipid-Coated NaLnF4 Nanoparticles: Demonstrating the Use of Lanthanide Nanoparticle-Based Reporters in Suspension and Imaging Mass Cytometry.

Lanthanide nanoparticles (LnNPs) have the potential to be used as high-sensitivity mass tag reporters in mass cytometry immunoassays. For this application, however, the LnNPs must be made colloidally stable in aqueous buffers, demonstrate minimal non-specific binding to cells, and have functional groups to attach antibodies or other targeting agents. One possible approach to address these requirements is by using lipid coating to modify the surface of the LnNPs. In this work, 39 nm diameter NaYF4:Yb, Er NPs (LnNPs) were coated with a lipid formulation consisting of egg sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium propane, cholesterol-(polyethylene glycol-600), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]. The resulting biotinylated lipid-coated LnNPs were characterized by dynamic light scattering to determine the hydrodynamic size and stability in phosphate buffered saline, and the composition of the lipid coatings was quantified by liquid chromatography-tandem mass spectrometry. The specific and non-specific binding of the biotinylated lipid-coated LnNPs to a model system of functionalized polystyrene microbeads were then tested by both suspension and imaging mass cytometry. We found that targeted binding with minimal non-specific binding can be achieved with the lipid-coated LnNPs and that the lipid composition of the coating has an impact on the performance of the LnNPs as mass cytometry reporters. These results additionally establish the importance of quantifying the composition of lipid-coated nanomaterials to optimize them more effectively for their desired application.

Scratching the Surface (Modification): Developing a Quantitative Liquid Chromatography-Tandem Mass Spectrometry Method for the Investigation of PEGylated and Non-PEGylated Lipid Mixtures on Lipid-Coated Lanthanide Nanoparticles.

We are interested in developing lanthanide nanoparticles (LnNPs) of the general formula NaLnF4 as high-sensitivity reagents for mass cytometry. These LnNPs must be coated to provide colloidal stability in aqueous buffer and functionality for detecting cellular biomarkers. Lipid bilayer coatings are a promising approach, but one requires an analytical technique to enable characterization of the NP coating composition as opposed to the composition of the lipid formulation used in the coating process. However, quantification of the lipid composition of lipid coatings on polymer and inorganic NPs is not common practice in the field. Here we describe a method based on high-performance liquid chromatography (LC) for separations and triple quadrupole tandem mass spectrometry (MS/MS) instrumentation for detection and show that it can quantify complex lipid mixtures using small (<1 µg) amounts of sample. Our lipid formulation consisted of a mixture of egg sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol-PEG600, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]. We were able to extract the coating from lipid-coated 35 nm diameter LnNPs, quantify the lipid/NP ratio, and show that the coating composition differed from the composition of the lipid formulation for several of the species. Knowledge of the actual composition of the lipid coating for lipid-coated NPs is critical for designing and optimizing application of these materials. Our results establish the value of LC-MS/MS characterization of lipid-coated NPs, thus providing an important new addition to the toolbox available for characterizing these types of nanomaterials.

In-Depth Analysis of the Effect of Fragmentation on the Crystallization-Driven Self-Assembly Growth Kinetics of 1D Micelles Studied by Seed Trapping.

Studying the growth of 1D structures formed by the self-assembly of crystalline-coil block copolymers in solution at elevated temperatures is a challenging task. Like most 1D fibril structures, they fragment and dissolve when the solution is heated, creating a mixture of surviving crystallites and free polymer chains. However, unlike protein fibrils, no new nuclei are formed upon cooling and only the surviving crystallites regrow. Here, we report how trapping these crystallites at elevated temperatures allowed us to study their growth kinetics at different annealing times and for different amounts of unimer added. We developed a model describing the growth kinetics of these crystallites that accounts for fragmentation accompanying the 1D growth process. We show that the growth kinetics follow a stretched exponential law that may be due to polymer fractionation. In addition, by evaluating the micelle growth rate as a function of the concentration of unimer present in solution, we could conclude that the micelle growth occurred in the mononucleation regime.

A Silica Coating Approach to Enhance Bioconjugation on Metal-Encoded Polystyrene Microbeads for Bead-Based Assays in Mass Cytometry.

Bead-based assays in flow cytometry are multiplexed analytical techniques that allow rapid and simultaneous detection and quantification of a large number of analytes from small volumes of samples. The development of corresponding bead-based assays in mass cytometry (MC) is highly desirable since it could increase the number of analytes detected in a single assay. The microbeads for these assays have to be labeled with metal isotopes for MC detection. One must also be able to functionalize the bead surface with affinity reagents to capture the analytes. Metal-encoded polystyrene microbeads prepared by multi-stage dispersion polymerization can produce effective isotopic signals in MC with relatively small bead-to-bead variations. However, functionalizing this microbead surface with bioaffinity agents remains challenging, possibly due to the interference of the steric-stabilizing PVP corona on the microbead surface. Here, we report a systematic investigation of a silica coating approach to coat Eu-encoded microbeads with thin silica shells, to functionalize the surface with amino groups, and to introduce bioaffinity agents. We examine the effect of silica shell roughness on the bioconjugation capacity and the effect of silica shell thickness on signal quality in MC measurements. To limit non-specific binding, we converted the amino groups on the microbead surface to carboxylic acid groups. Antibodies were effectively attached to microbead by first conjugating NeutrAvidin to the carboxyl-modified bead surface and then attaching biotinylated antibodies to the NeutrAvidin-modified bead surface. The antibody-modified microbeads can specifically capture antigens, which were marked with isotopic labels, and generate strong signals in MC. These are promising results for the development of bead-based assays in MC.

Site-Specific Conjugation of Metal-Chelating Polymers to Anti-Frizzled-2 Antibodies via Microbial Transglutaminase.

Metal-chelating polymer-based radioimmunoconjugates (RICs) are effective agents for radioimmunotherapy but are currently limited by nonspecific binding and off-target organ uptake. Nonspecific binding appears after conjugation of the polymer to the antibody and may be related to random lysine conjugation since the polymers themselves do not bind to cells. To investigate the role of conjugation sites on nonspecific binding of polymer RICs, we developed a microbial transglutaminase reaction to prepare site-specific antibody-polymer conjugates. The reaction was enabled by introducing a Q-tag (i.e., 7M48) into antibody (i.e., Fab) fragments and synthesizing a polyglutamide-based metal-chelating polymer with a PEG amine block to yield substrates. Mass spectrometric analyses confirmed that the microbial transglutaminase conjugation reaction was site-specific. For comparison, random lysine conjugation analogs with an average of one polymer per Fab were prepared by bis-aryl hydrazone conjugation. Conjugates were prepared from an anti-frizzled-2 Fab to target the Wnt pathway, along with a nonbinding specificity control, anti-Luciferase Fab. Fabs were engineered from a trastuzumab-based IgG1 framework and lack lysines in the antigen-binding site. Conjugates were analyzed for thermal conformational stability by differential scanning fluorimetry, which showed that the site-specific conjugate had a similar melting temperature to the parent Fab. Binding assays by biolayer interferometry showed that the site-specific anti-frizzled-2 conjugate maintained high affinity to the antigen, while the random conjugate showed a 10-fold decrease in affinity, which was largely due to changes in association rates. Radioligand cell-binding assays on frizzled-2+ PANC-1 cells and frizzled-2- CHO cells showed that the site-specific anti-frizzled-2 conjugate had ca. 4-fold lower nonspecific binding compared to the random conjugate. Site-specific conjugation appeared to reduce nonspecific binding associated with random conjugation of the polymer in polymer RICs.

Uniform 1D Micelles and Patchy & Block Comicelles via Scalable, One-Step Crystallization-Driven Block Copolymer Self-Assembly.

Fiber-like (1D) core-crystalline micelles of uniform length can be obtained in protocols involving multiple steps from block copolymers (BCPs) in which crystallization of the core-forming polymer drives the self-assembly. Here we report a systematic study that shows that adding small amounts (<5 w/w%) of a homopolymer corresponding to the core-forming block of the BCP enables uniform 1D micelles (mean lengths Ln = 0.6 to 9.7 µm) to be obtained in a single step, simply by heating the mixture in a selective solvent followed by slow cooling. A series of poly(ferrocenyldimethylsilane) (PFS) BCPs with different corona-forming blocks and different compositions as well as PFS homopolymers of different lengths were examined. Dye labeling and confocal fluorescence microscopy showed that the homopolymer ends up in the center of the micelle, signaling that it served as the initial seed for epitaxial micelle growth. The rate of unimer addition was strongly enhanced by the length of the PFS block, and this enabled more complex structures to be formed in one-pot self-assembly experiments from mixtures of two or three BCPs with different PFS block lengths. Furthermore, BCP mixtures that included PFS-b-PI (PI = polyisoprene) and PFS-b-PDMS with similar PFS block lengths resulted in simultaneous addition to growing micelles, resulting in a patchy block that could be visualized by staining the vinyl groups of the PI with Pt nanoparticles. This approach also enabled scale up, so that uniform 1D micelles of controlled architecture can be obtained at concentrations of 10 w/w % solids or more.

Influence of the Sodium Precursor on the Cubic-to-Hexagonal Phase Transformation and Controlled Preparation of Uniform NaNdF4 Nanoparticles.

NaLnF4 nanoparticles (NPs) with lighter lanthanides (where Ln = La, Ce, Nd, or Pr) are more difficult to prepare than those with heavier lanthanides [Naduviledathu et al. Chem Mater., 2014, 26, 5689]. Our knowledge is weakest for NaLnF4 NPs with the lowest atomic mass lanthanides (Yan's group 1: La to Nd) and more advanced for group 2 (Sm to Tb) NaLnF4 NPs [Mai et al., J. Am. Chem. Soc., 2006, 128, 6426]. Here we focus on the synthesis of NaNdF4 NPs. We employed the high-temperature chemical coprecipitation method and explored the influence of a wide range of synthesis parameters (e.g., reaction time and temperature, precursor ratios (Na+/Nd3+ and F-/Nd3+), choice of a sodium precursor (Na-oleate or NaOH), and the amount of oleic acid) on the size and uniformity of the NPs obtained. We tried to identify "sweet spots" in the reaction space that led to uniform NaNdF4 NPs with sizes appropriate for mass tag applications in mass cytometry. We were able to obtain NPs with a variety of sizes in the range of 5-38 nm with several different shapes (e.g., polyhedra, spheres, and rods). XRD patterns recorded for aliquots collected at different reaction time intervals revealed that NaNdF4 nucleated in the cubic phase (α) and then transformed to the hexagonal phase (ß) as the reaction progressed up to 2 h. A very striking observation was that the NPs synthesized using NaOH as a reactant preferred to remain in the α-phase, and for a lower reaction temperature (285 °C), did not undergo a phase transformation to the ß-phase over 2 h of reaction time. Under similar experimental conditions, NPs prepared using Na-oleate exhibited an α → ß phase transformation. Nevertheless, NaNdF4 NPs prepared at a higher temperature (315 °C) using either of the Na+ precursors exhibited the α → ß phase transformation over time. This transition, however, appeared to be faster in the case of the NPs synthesized using Na-oleate. We found that, in many instances, syntheses carried out using Na-oleate produced more uniform NPs compared to those synthesized using NaOH. Under the conditions we employed for the Na-oleate precursor, the NPs initially formed were polydisperse spheres that evolved into irregular polyhedra and eventually formed more uniform rod-shaped NPs. The aspect ratio of the final NPs depended on the Na+/Nd3+ precursor ratio. High-resolution transmission electron micrographs and corresponding fast Fourier transform of the data provided information about the preferred growth direction of the NaNdF4 nanorods.