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Mass cytometry (MC) measures metal isotope signals from single cells and bead samples. Since large numbers of isotopes can be employed as labels, mass cytometry is a powerful analytical technique for multiparameter cytometric assays. The calibration protocol in MC is a critical algorithm, which employs metal-encoded microbeads as an internal standard to correct the data for instrumental signal drift. The current generation of commercially available beads carries four lanthanide elements (cerium, europium, holmium, and lutetium). However, this is not sufficient to calibrate the full span of detection channels, ranging from yttrium (89 amu) to bismuth (209 amu), which are now available. To address this issue we prepared polystyrene microbeads encoded with seven elements (yttrium, indium, and bismuth in addition to the four lanthanides) by multistage dispersion polymerization for MC calibration and normalization. The bead synthesis conditions were optimized to obtain microbeads that were uniform in size and generated strong MC signal intensities at similar levels for the eight encoded isotopes. Metal ion leaching from the beads under storage and application conditions was also examined. We demonstrated that the precision of normalized MC signals in the MC detection channels was improved by employing seven-element-encoded microbeads as a standard.

Although there have been many reports on the synthesis of NaLnF4nanoparticles (NPs), in particular ones that show upconversion luminescence, there is still an insufficient understanding of the mechanism to design the set of reaction conditions that will result in NPs of a particular diameter with a narrow size distribution. Here, we describe experiments that lead to the synthesis of uniform NaLnF4(Ln = Sm, Eu, Tb, Ho) nanoparticles (NPs) with diameters between 4 and 30 nm. NPs, in this size range, are particularly important as potential reagents for mass cytometry. The effects of varying the amount of ligand (oleic acid), precursor ratios, heating time, and temperature are discussed. In general, we found that identical reaction conditions cannot be used to synthesize NPs of identical sizes for different lanthanide elements. We found that to obtain larger uniform NaLnF4NPs, conditions should be found such that the NPs initially nucleate in the cubic phase and undergo a subsequent phase change to the hexagonal phase. The nucleating phase was found to be affected by the Na/Ln and F/Ln precursor ratios, where larger ratios led to initial nucleation in the hexagonal phase and smaller ratios led to initial nucleation in the cubic phase and progressively larger NPs. The nucleating phase was also affected by the amount of the ligand; however, the trends we observed were not consistent between different lanthanides.

Nucleated self-assembly in selective solvents of core-crystalline block copolymers (BCPs) is a special case of living supramolecular polymerization, leading to rodlike micelles of controlled and uniform length. For the crystallization-driven self-assembly of PFS-containing BCPs (PFS = polyferrocenyldimethylsilane), the formation of block comicelles by sequential addition of different BCPs is well-established. But there are only a few examples of living copolymerization, the simultaneous addition of pairs of BCPs with different corona-forming chains. At present, relatively little is known about the competitive kinetics of different BCPs crystallizing on a common seed. Here we report a systematic study of the competitive seeded growth kinetics of pairs of linear PFS-containing BCPs and show that one can manipulate the kinetics to control the morphology of the comicelles. We found that the seeded-growth kinetics of the individual BCP unimer dominates the coassembly behavior and thus the morphology of the corona. Patchy comicelles with microphase-segregated corona chains are formed when the epitaxial growth rates of the two different BCPs on the common seed are similar. In contrast, factors that lead to dissimilar growth rates (long corona-forming blocks or introduction of charges on corona-forming chains) promote large-scale separation of the corona blocks, leading to block comicelles. Because the termini of the comicelles remain living, they can further direct the growth of unimers, resulting in hierarchical block comicelles with patchy blocks and single-component (homo) blocks. Furthermore, the patchy comicelles can be loaded with either gold or platinum nanoparticles, generating organic–inorganic hybrid materials with potential application in catalysis.

A metal-chelating polymer (MCP) with a polyglutamide (PGlu) backbone presenting on average 13 DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators for complexing 111In or 177Lu and 10 polyethylene glycol (PEG) chains to minimize liver and spleen uptake was conjugated to antiepidermal growth factor receptor (EGFR) monoclonal antibody (mAb), panitumumab. Because panitumumab-MCP may be dual-labeled with 111In and 177Lu for SPECT, or radioimmunotherapy (RIT) exploiting the Auger electrons or β-particle emissions, respectively, we propose that panitumumab-MCP could be a useful theranostic agent for EGFR-positive PnCa. Bioconjugation was achieved by reaction of a hydrazine nicotinamide (HyNIC) group on the MCP with an aryl aromatic aldehyde introduced into panitumumab by reaction with succinimidyl-4-formylbenzamide (S-4FB). The conjugation reaction was monitored by measurement of the chromophoric bis-aryl hydrazone bond formed (ε350 nm= 24 500 M–1cm–1) to achieve two MCPs/panitumumab. Labeling of panitumumab-MCP with 111In or 177Lu demonstrated that masses as small as 0.1 μg were labeled to >90% labeling efficiency (L.E.) and a specific activity (SA) of >70 MBq/μg. Panitumumab-DOTA incorporating two DOTA per mAb was labeled with 111In or 177Lu to a maximum SA of 65 MBq/μg and 46 MBq/μg, respectively. Panitumumab-MCP-177Lu exhibited saturable binding to EGFR-overexpressing MDA-MB-468 human breast cancer cells. The Kdfor binding of panitumumab-MCP-177Lu to EGFR (2.2 ± 0.6 nmol/L) was not significantly different than panitumumab-DOTA-177Lu (1.0 ± 0.4 nmol/L). 111In and 177Lu were stably complexed to panitumumab-MCP. Panitumumab-MCP-111In exhibited similar whole body retention (55–60%) as panitumumab-DOTA-111In in NOD-scid mice up to 72 h postinjection (p.i.) and equivalent excretion of radioactivity into the urine and feces. The uptake of panitumumab-MCP-111In in most normal tissues in NOD-scid mice with EGFR-positive PANC-1 human pancreatic cancer (PnCa) xenografts at 72 h p.i. was not significantly different than panitumumab-DOTA-111In, except for the liver which was 3-fold greater for panitumumab-MCP-111In. Tumor uptake of panitumumab-MCP-111In (6.9 ± 1.3%ID/g) was not significantly different than panitumumab-DOTA-11In (6.6 ± 3.3%ID/g). Tumor uptake of panitumumab-MCP-111In and panitumumab-DOTA-111In were reduced by preadministration of excess panitumumab, suggesting EGFR-mediated uptake. Tumor uptake of nonspecific IgG-MCP (5.4 ± 0.3%ID/g) was unexpectedly similar to panitumumab-MCP-111In. An increased hydrodynamic radius of IgG when conjugated to an MCP may encourage tumor uptake via the enhanced permeability and retention (EPR) effect. Tumor uptake of panitumumab-DOTA-111In was 3.5-fold significantly higher than IgG-DOTA-111In. PANC-1 tumors were imaged by microSPECT/CT at 72 h p.i. of panitumumab-MCP-111In or panitumumab-DOTA-111In. Tumors were not visualized with preadministration of excess panitumumab to block EGFR, or with nonspecific IgG radioimmunoconjugates. We conclude that linking panitumumab to an MCP enabled higher SA labeling with 111In and 177Lu than DOTA-conjugated panitumumab, with preserved EGFR binding in vitro and comparable tumor localization in vivo in mice with s.c. PANC-1 human PnCa xenografts. Normal tissue distribution was similar except for the liver which was higher for the polymer radioimmunoconjugates.

In recent years, there has been an explosion in the number of papers dealing with “cylindrical” PS-b-PMMA, for nanoscopic template or similar applications. This paper deals with two aspects of these systems. One is the detailed shape of the cores, i.e., the degree to which they are actually cylindrical in the hexagonal unit cell, as well as the nature of the core–corona interphase region. The second one relates to the use of fluorescence techniques to study these interphases, including whether or not one needs to consider energy transfer from one cylinder to neighboring ones. We approached these questions with numerical self-consistent field theory plus simulations of energy transfer based on them, and we examined two systems: one which mimics “typical” experimental systems at weak to moderate segregation, and one chosen to explore the limits of very weak segregation and polymer composition where the effects of interest could be maximal. In addition to providing basic information about the system characteristics, the results provide practical guidance on using the assumptions of cylindrical symmetry and no intercell energy transfer.

Thermoresponsive colloidal hydrogels (aqueous microgels) made from poly[oligo(ethylene glycol) methacrylate] (PEGMA) are an interesting class of biomaterials due to their sharp thermal transition and excellent biocompatibility. However, the inherent protein repellency of PEGMA has made the biofunctionalization of these microgels difficult and prevented them from reaching their full potential in applications such as a protein carrier. Here, we report the synthesis of thermoresponsive PEGMA microgels and the covalent attachment of horseradish peroxidase (HRP, as a model protein) to these microgels. We prepared our microgels by the precipitation copolymerization of 70 mol % OEGMA300and 30 mol % methacrylic acid in water. The resulting microgels showed a volume phase transition temperature (VPTT) of ca. 60 °C in acidic buffers and in DI water but in neutral and basic buffers did not show a thermal response up to 75 °C. The direct immobilization of HRP to the activated carboxylic groups in the microgel backbone was unsuccessful, but using a diamine spacer with four ethylene glycol units, we were able to covalently attach this enzyme to our PEGMA microgels through bis-aryl hydrazone chemistry. HRP has its maximum activity at ca. 50 °C. At higher temperatures, the activity was reduced, but the microgel-bound enzyme showed less reduction in activity than the native enzyme and no change in activity associated with the VPTT. In addition, PEGMA microgels stabilized the attached enzymes against thermal denaturation. For example, our results showed that the enzyme immobilized on the PEGMA microgel lost its activity 3.4 times slower than the free enzyme in the first 5 h of annealing at 50 °C. The bioconjugation strategy introduced here could serve as a model for the covalent attachment of other biomacromolecules to the protein-repellent PEGMA microgels.

Our aim was to evaluate the effectiveness and normal tissue toxicity of radioimmunotherapy (RIT) of s.c. PANC-1 human pancreatic cancer (PnCa) xenografts in NRG mice using anti-EGFR panitumumab linked to metal-chelating polymers (MCPs) that present 13 DOTA chelators to complex the β-emitter, 177Lu. The clonogenic survival (CS) of PANC-1 cells treated in vitro with panitumumab-MCP-177Lu (0.3–1.2 MBq) and DNA double-strand breaks (DSBs) in the nucleus of these cells were measured by confocal immunofluorescence microscopy for γ-H2AX. Subcellular distribution of radioactivity for panitumumab-MCP-177Lu was measured, and absorbed doses to the cell nucleus were calculated. Normal tissue toxicity was assessed in non tumor-bearing NRG mice by monitoring body weight, complete blood cell counts (CBC), serum alanine aminotransferase (ALT), and creatinine (Cr) after i.v. injection of 6 MBq (10 μg) of panitumumab-MCP-177Lu. RIT was performed in NRG mice with s.c. PANC-1 tumors injected i.v. with 6 MBq (10 μg) of panitumumab-MCP-177Lu. Control mice received nonspecific human IgG-MCP-177Lu (6 MBq; 10 μg), unlabeled panitumumab (10 μg), or normal saline. The tumor growth index (TGI) was compared. Tumor and normal organ doses were estimated based on biodistribution studies. Panitumumab-MCP-177Lu reduced the CS of PANC-1 cells in vitro by 7.7-fold at the highest amount tested (1.2 MBq). Unlabeled panitumumab had no effect on the CS of PANC-1 cells. γ-H2AX foci were increased by 3.8-fold by panitumumab-MCP-177Lu. Panitumumab-MCP-177Lu deposited 3.84 Gy in the nucleus of PANC-1 cells. Administration of panitumumab-MCP-177Lu (6 MBq; 10 μg) to NRG mice caused no change in body weight, CBC, or ALT and only a slight increase in Cr compared to NRG mice treated with normal saline. Panitumumab-MCP-177Lu strongly inhibited tumor growth in NRG mice (TGI = 2.3 ± 0.2) compared to normal saline-treated mice (TGI = 5.8 ± 0.5; P< 0.01). Unlabeled panitumumab had no effect on tumor growth (TGI = 6.0 ± 1.6; P> 0.05). The absorbed dose of PANC-1 tumors was 12.3 Gy. The highest normal organ doses were absorbed by the pancreas, liver, spleen, and kidneys. We conclude that EGFR-targeted RIT with panitumumab-MCP-177Lu was able to overcome resistance to panitumumab in KRAS mutant PANC-1 tumors in NRG mice and may be a promising approach to treatment of PnCa in humans.

Patrick Pinenq [+] Mitchell A. Winnik [**] University of Toronto [*] Didier Juhue [+] ELF-Atochem CERDATO [++] INTRODUCTION This paper describes factors affecting polymer diffusion in films prepared from aqueous [...]