Metal-chelating Polymers Research Articles

Optimizing the Structure of a Pt Metal-Chelating Polymer to Reduce Nonspecific Binding for Mass Cytometry.

Mass cytometry is a bioanalytic tool based on atomic mass spectrometry for detecting biomarker expression on individual cells. Current reagents employ metal-chelating polymers binding isotopes of hard metal ions. Polymers bearing chelators for soft metal ions offer the promise for a large increase in multiplexing capabilities, but examples reported so far often have unacceptably high levels of nonspecific binding (NSB). We recently reported a new class of metal-chelating polymers with dipicolylamine (DPA) chelators that could bind Re and Pt. They also showed significant levels of NSB. Here, to reduce the NSB of the Pt-DPA polymer, we grafted water-soluble oligomers to the distal end of the dipicolylamine pendant group. Methoxy(polyethylene glycol) (DP = 24) was effective as was poly(sulfobetaine methacrylate) (DP = 29). Reacting the Pt-Cl bond of the metalated polymer with glutathione was remarkably effective at suppressing NSB. These results open the door to Pt-isotope-based metal-chelating polymers as new mass tags for mass cytometry.

Synthesis, Characterization, and Applications of a Superior Dendrimer-Based Polymer for Multiplexed Ion Beam Imaging Time-of-Flight Analysis.

High-dimensional single-cell mass spectrometric imaging techniques such as multiplexed ion beam imaging by time-of-flight mass spectrometry (MIBI-TOF), imaging mass cytometry (IMC), and flow cytometry-based CyTOF utilize antibodies conjugated to linear metal-chelating polymers. Here, we report on the synthesis and characterization of a dendrimer-based polymer and its utilization in tissue imaging using MIBI-TOF. We compared the staining performance in FFPE tissue of antibodies for lineage-specific immune proteins (CD20, CD3, CD45, FoxP3) that were conjugated with dendrimer or linear polymer. Staining of serial tissue sections with dendron-conjugated and linear-polymer-conjugated antibodies revealed comparable avidities of dendrons and linear polymers with log2 (ratio of mean positive pixel intensity of staining for linear polymers to dendrons) within the range ±0.25. Interestingly, dendron-conjugated antibodies were observed to have some advantages over linear polymer-conjugated antibodies. For example, tissue staining of a nuclear protein, FoxP3 with dendron-conjugated antibodies showed notably less background staining than that of linear-polymer-conjugated antibodies. Additionally, dendron-conjugated antibodies did not exhibit off-target cytosolic binding in neural tissue typically observed when using linear polymer conjugates. Taken together, this work provides a versatile framework for using third-generation dendron-conjugated antibodies with improved staining over conventional linear polymers.

A universal mass tag based on polystyrene nanoparticles for single-cell multiplexing with mass cytometry

Mass cytometry (MC) is an emerging bioanalytical technique for high-dimensional biomarkers interrogation simultaneously on individual cells. However, the sensitivity and multiplexed analysis ability of MC was highly restricted by the current metal chelating polymer (MCP) mass tags. Herein, a new design strategy for MC mass tags by using a commercial available and low cost classical material, polystyrene nanoparticle (PS-NP) to carry metals was reported. Unlike inorganic materials, sub-micron-grade metal-loaded polystyrene can be easily detected by MC, thus it is not essential to pursue extremely small particle size in this mass tag design strategy. An altered cell staining buffer can significantly lower the nonspecific binding (NSB) of non-functionalized PS-NPs, revealing another method to lower NSB beside surface modification. The metal doped PS-NP_Abs mass tags showed high compatibility with MCP mass tags and 5-fold higher sensitivity. By using Hf doped PS-NP_Abs as mass tags, four new MC detection channels (177Hf, 178Hf, 179Hf and 180Hf) were developed. In general, this work provides a new strategy in designing MC mass tags and lowering NSB, opening up possibility of introducing more potential MC mass tag candidates.

Characterizing Ion-Polymer Interactions in Aqueous Environment with Electric Fields.

Polymers make the basis of highly tunable materials that could be designed and optimized for metal recovery from aqueous environments. While experimental studies show that this approach has potential, it suffers from a limited knowledge of the detailed molecular interaction between polymers and target metal ions. Here, we propose to calculate intrinsic electric fields from polarizable force field molecular dynamics simulations to characterize the driving force behind Eu3+ motion in the presence of poly(ethylenimine methylenephosphonate), a specifically designed metal chelating polymer. Focusing on the metal chelation initiation step (i.e., before binding), we can rationalize the role of each molecule on ion dynamics by projecting these electric fields along the direction of ion motion. We find that the polymer functional groups act indirectly, and the polymer-metal ion interaction is actually mediated by water. This result is consistent with the experimental observation that metal sequestration by these polymers is entropically driven. This study suggests that electric field calculations can help the design of metal chelating polymers, for example, by seeking to optimize polymer-solvent interactions rather than polymer-ion interactions.

Chemical activation of commodity plastics for patterned electroless deposition of robust metallic films.

A general approach to increase the adhesion of metal films to commodity plastic substrates using a metal-chelating polymer, polyethyleneimine, in conjunction with patterned electroless deposition is described. This general fabrication method is compatible with a diverse array of plastics and metals with properties applicable to flexible electronic circuits and electrochemical cells.

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.

Improving the Stability of Non-Noble-Metal M-N-C Catalysts for Proton-Exchange-Membrane Fuel Cells through M-N Bond Length and Coordination Regulation.

An effective and universal strategy is developed to enhance the stability of the non-noble-metal M-Nx /C catalyst in proton exchange membrane fuel cells (PEMFCs) by improving the bonding strength between metal ions and chelating polymers, i.e., poly(acrylic acid) (PAA) homopolymer and poly(acrylic acid-maleic acid) (P(AA-MA)) copolymer with different AA/MA ratios. Mössbauer spectroscopy and X-ray absorption spectroscopy (XAS) reveal that the optimal P(AA-MA)-Fe-N catalyst with a higher Fe3+ -polymer binding constant possesses longer FeN bonds and exclusive Fe-N4 /C moiety compared to PAA-Fe-N, which consists of ≈15% low-coordinated Fe-N2 /N3 structures. The optimized P(AA-MA)-Fe-N catalyst exhibits outstanding ORR activity and stability in both half-cell and PEMFC cathodes, with the retention rate of current density approaching 100% for the first 37 h at 0.55V in an H2 -air fuel cell. Density functional theory (DFT) calculations suggest that the Fe-N4 /C site could optimize the difference between the adsorption energy of the Fe atoms on the support (Ead ) and the bulk cohesive energy (Ecoh ) relative to Fe-N2 /N3 moieties, thereby strongly stabilizing Fe centers against demetalation.

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.

Effect of Copolymer Structure on Rare-Earth-Element Chelation Thermodynamics.

Rare-earth elements (REEs) are crucial to modern technology, leading to a high demand for materials capable of REE extraction and purification. Metal-chelating polymers (e.g., polycarboxylic acids, polyamines, and others) are particularly useful in these applications due to their synthetic accessibility and high selectivity. Copolymers with varied mole fractions of acrylic acid and methyl acrylate are synthesized and isothermal titration calorimetry (ITC) to measure the thermodynamics of REE binding for each material is used. Across a series of copolymer compositions, entropically driven binding thermodynamics (∆G, ∆H, and ∆S) that appear to be independent of χAcrylic Acid are found. ITC stoichiometry data reveal that each copolymer requires between four and five repeat units to bind each REE. These data suggest that alterations in the copolymer structure do not affect the overall binding thermodynamics of REEs to these copolymers.

Mass Cytometry Tags: Where Chemistry Meets Single-Cell Analysis.

Mass cytometry is a highly multiparametric proteomic technology that allows the measurement and quantification of nearly 50 markers with single-cell resolution. Mass cytometry reagents are probes tagged with metal isotopes of defined mass and act as reporters. Metals are detected using inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS). Many different types of mass-tag reagents have been developed to afford myriad applications. We have classified these compounds into polymer-based mass-tag reagents, nonpolymer-based mass-tag reagents, and inorganic nanoparticles. Metal-chelating polymers (MCPs) are widely used to profile and quantify cellular biomarkers; however, both the range of metals that can be detected and the metal signals have to be improved. Several strategies such as the inclusion of chelating agents or highly branched polymers may overcome these issues. Biocompatible materials such as polystyrene and inorganic nanoparticles are also of profound interest in mass cytometry. While polystyrene allows the inclusion of a wide variety of metals, the high metal content of inorganic nanoparticles offers an excellent opportunity to increase the signal from the metals to detect low-abundance biomarkers. Nonpolymer-based mass-tag reagents offer multiple applications: cell detection, cell cycle property determination, biomarker detection, and mass-tag cellular barcoding (MCB). Recent developments have been achieved in live cell barcoding by targeting proteins (CD45, b2m, and CD298), by using small and nonpolar probes or by ratiometric barcoding. From this perspective, the principal applications, strengths, and shortcomings of mass-tag reagents are highlighted, summarized, and discussed, with special emphasis on mass-tag reagents for MCB. Thereafter, the future perspectives of mass-tag reagents are discussed considering the current state-of-the-art technologies.

Radioimmunotherapy of PANC-1 human pancreatic cancer xenografts in NOD/SCID or NRG mice with Panitumumab labeled with Auger electron emitting, 111In or \u03b2-particle emitting, 177Lu

BackgroundEpidermal growth factor receptors (EGFR) are overexpressed on > 90% of pancreatic cancers (PnCa) and represent an attractive target for the development of novel therapies, including radioimmunotherapy (RIT). Our aim was to study RIT of subcutaneous (s.c.) PANC-1 human PnCa xenografts in mice using the anti-EGFR monoclonal antibody, panitumumab labeled with Auger electron (AE)-emitting, 111In or β-particle emitting, 177Lu at amounts that were non-toxic to normal tissues.ResultsPanitumumab was conjugated to DOTA chelators for complexing 111In or 177Lu (panitumumab-DOTA-[111In]In and panitumumab-DOTA-[177Lu]Lu) or to a metal-chelating polymer (MCP) with multiple DOTA to bind 111In (panitumumab-MCP-[111In]In). Panitumumab-DOTA-[177Lu]Lu was more effective per MBq exposure at reducing the clonogenic survival in vitro of PANC-1 cells than panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In. Panitumumab-DOTA-[177Lu]Lu caused the greatest density of DNA double-strand breaks (DSBs) in the nucleus measured by immunofluorescence for γ-H2AX. The absorbed dose in the nucleus was 3.9-fold higher for panitumumab-DOTA-[177Lu]Lu than panitumumab-DOTA-[111In]In and 7.7-fold greater than panitumumab-MCP-[111In]In. No normal tissue toxicity was observed in NOD/SCID mice injected intravenously (i.v.) with 10.0 MBq (10 μg; ~ 0.07 nmoles) of panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In or in NRG mice injected i.v. with 6.0 MBq (10 μg; ~ 0.07 nmoles) of panitumumab-DOTA-[177Lu]Lu. There was no decrease in complete blood cell counts (CBC) or increased serum alanine aminotransferase (ALT) or creatinine (Cr) or decreased body weight. RIT inhibited the growth of PANC-1 tumours but a 5-fold greater total amount of panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In (30 MBq; 30 μg; ~ 0.21 nmoles) administered in three fractionated amounts every three weeks was required to achieve greater or equivalent tumour growth inhibition, respectively, compared to a single amount of panitumumab-DOTA-[177Lu]Lu (6 MBq; 10 μg; ~ 0.07 nmoles). The tumour doubling time (TDT) for NOD/SCID mice with s.c. PANC-1 tumours treated with panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In was 51.8 days and 28.1 days, respectively. Panitumumab was ineffective yielding a TDT of 15.3 days vs. 15.6 days for normal saline treated mice. RIT of NRG mice with s.c. PANC-1 tumours with 6.0 MBq (10 μg; ~ 0.07 nmoles) of panitumumab-DOTA-[177Lu]Lu increased the TDT to 20.9 days vs. 11.5 days for panitumumab and 9.1 days for normal saline. The absorbed doses in PANC-1 tumours were 8.8 ± 3.0 Gy and 2.6 ± 0.3 Gy for panitumumab-DOTA-[111In]In and panitumumab-MCP-[111In]In, respectively, and 11.6 ± 4.9 Gy for panitumumab-DOTA-[177Lu]Lu.ConclusionRIT with panitumumab labeled with Auger electron-emitting, 111In or β-particle-emitting, 177Lu inhibited the growth of s.c. PANC-1 tumours in NOD/SCID or NRG mice, at administered amounts that caused no normal tissue toxicity. We conclude that EGFR-targeted RIT is a promising approach to treatment of PnCa.

Tantalum Oxide Nanoparticle-Based Mass Tag for Mass Cytometry.

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.

Synthesis of a metal-chelating polymer with NOTA pendants as a carrier for 64Cu, intended for radioimmunotherapy

As a positron emitter, 64Cu is useful for positron emission tomography. It can also generate Auger electrons that are useful for therapy. Many radioimmunoconjugates labelled with 64Cu involve covalent attachment of bifunctional chelators directly to the antibody (Ab) of interest. To increase its therapeutic potential, a metal-chelating polymer (MCP) with multiple chelators can be conjugated the Ab. In this way, one can incorporate multiple 64Cu ions per Ab. Here we describe the synthesis of a MCP designed for attachment to trastuzumab (tmab), an antibody (Ab) used to treat breast cancer patients with tumours that overexpress human epidermal growth factor receptor 2. We chose 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) chelator to complex with 64Cu, and in our design, the polymer would carry multiple copies of the NOTA chelator as pendant groups. The Cu2+/NOTA complex has a net negative charge. We began by using RAFT polymerization to synthesize an end-functional poly(2-aminoethyl acrylamide) polymer, in which we could use the amino groups to attach the chelator and other substituents. In a previous study (Biomacromolecules. 13 (2012) 2831–2842), we found that MCP radioimmunoconjugates with a negative charge in the repeat unit led to high liver up-take in a mouse model. Thus, we chose to introduce ca. 50% methoxy-dodecaethylene glycol (mPEG12) pendant groups to confer “stealth”. As a control, we also synthesized a similar polymer with ca. 50% methoxyethyl pendants (EO1). NOTA chelators were attached to remaining pendant groups. We then end-capped the MCPs with succinic acid, radiolabeled the MCPs with 64Cu and injected the radiolabeled MCPs into healthy Balb/c mice via the tail vein. We observed that a large amount of the MCPs were retained in the kidneys. We discuss what properties of the MCPs may have contributed to its accumulation in the kidneys.

An MRI contrast agent based on a zwitterionic metal-chelating polymer for hepatorenal angiography and tumor imaging.

MRI contrast agents such as paramagnetic Gd(iii)-chelates, can improve the ability of MRI in differentiating diseased and healthy tissues, and have been widely used in clinical diagnosis. However, the enhancement effect of small molecular MRI contrast agents is unsatisfied due to their relative high rotation rates. Furthermore, the small molecular contrast agents also suffer from the short blood half-life and nonspecific extracellular diffusion in tissues, which also restricts their applications. To address these issues, we developed a macromolecular MRI contrast agent based on a zwitterionic metal-chelating polymer. Poly(acrylic acid) (PAA) was chosen as the main chain, and diethylenetriamine pentaacetic acid (DTPA) as the metal-chelating group was coupled through the carboxyl groups of PAA using diethylenetriamine (DET) as a linker. The macromolecular MRI contrast agent constructed by chelating with Gd3+ (Gd-PAA) exhibited a much higher longitudinal relaxation rate (r1) than the clinical contrast agent Gd-DTPA. Importantly, due to the stealth ability of the zwitterionic structure, Gd-PAA can reside in the blood long enough without any microvascular leakage in the extracellular space of normal tissues, which allows it to be used for precise blood MR imaging, such as hepatorenal angiography, but also for tumor imaging because of the enhanced permeability and retention (EPR) effecta. Besides, the result of long-term toxicity tests highlights the safety feature of the current contrast agent. Hence, the current contrast agent overcomes the defect of traditional small molecular Gd(iii)-based T1-weighted contrast agents and shows great prospects for future clinical applications.

Metal Chelating Polymer Thin Films by Surface-Initiated ROMP and Modification

We report the surface-initiated ring-opening metathesis polymerization (SiROMP) of hydroxamic acid-containing, metal-chelating polymer thin films. SiROMP of trans-5-norbornene-2,3-dicarbonyl chloride (NBDAC) is introduced as a versatile platform to achieve many functional polymer films via simple exposure of pNBDAC films to reagents. This modification strategy owes its success to the fast and high-yield reaction of acyl chlorides with alcohols, amines, water, and other molecules. The polymerization of NBDAC is performed with monomer in the vapor phase and exhibits rapid kinetics, producing ∼400 nm thick films within 5 min. Because of the high reactivity of the acyl chloride groups in the film, the pNBDAC films are easily modified to produce side chains of carboxylic acids, esters, and amides via the reaction of the acyl group with water, alcohols, and amines, respectively. Exposure of the pNBDAC film to hydroxylamine results in a film with hydroxamic acid functionality that is capable of chelating various metal ions. The chelation process effectively cross-links the polymer chains to greatly improve the thermal and pH stability of the film in solution, elevate resistance against water and ion transfer, and alter the mechanical properties to a more solid-like film.

A metal-chelating polymer for chelating zirconium and its use in mass cytometry

Mass cytometry (MC) uses antibodies (Abs) labeled with heavy metal isotopes to monitor biomarker expression on individual cells. Metal-chelating polymers (MCPs) conjugated to the Abs increase the number of metal ions carried per Ab, and this increases the signal generated per Ab. Although the MC detector can detect 134 metal isotopes with unique weight simultaneously, in the current literature a maximum of 43 isotopes have been analyzed simultaneously. Currently available MCPs for MC are well-suited for use with lanthanides and bismuth, but are incompatible with other metals of interest. Here we examine a new MCP with deferoxamine pendant groups designed to chelate Zr ions. Zr has 4 isotopes useful for MC. To overcome the limited water solubility of deferoxamine groups, we introduced pendant polyethylene glycol chains. The polymer also included fluorescein pendant groups, along with a biotin terminal end-group. Once loaded with Zr, the polymers were incubated with streptavidin-coated polystyrene microbead and analyzed using epifluorescence microscopy and imaging MC.

Lanthanide nanoparticles for high sensitivity multiparameter single cell analysis.

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 via 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 NaHoF4 NPs (d ∼ 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.

Method for Tagging Antibodies with Metals for Mass Cytometry Experiments.

Mass cytometers are time-of-flight (TOF) mass spectrometer-coupled flow cytometers (known as CyTOFs) that quantify the abundance of metal-tagged antibodies (Abs) or other cellular probes within single cell suspensions or laser-ablated tissue sections. While many strategies exist for covalently crosslinking to proteins, the Fluidigm MaxPar® process is currently the most widely used and involves first loading a metal-chelating polymer with an elementally and isotopically enriched metal. Once the chelation sites have been filled, a maleimide moiety on the polymer is reacted with the free thiol groups on the partially reduced monoclonal immunoglobulin G (IgG) Ab to form an irreversible covalent bond. Here we describe modifications to the Fluidigm MaxPar® protocol that increase the quantity of Ab per reaction up to 150μg, introduce an initial Ab quality control step, utilize metal labels not included in the Fluidigm catalog, and provide an option to perform two reactions in one centrifugal filter.

Radioimmunotherapy of PANC-1 Human Pancreatic Cancer Xenografts in NRG Mice with Panitumumab Modified with Metal-Chelating Polymers Complexed to 177Lu.

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.

PH-Responsive fluorescent dye-labeled metal-chelating polymer with embedded cadmium telluride quantum dots for controlled drug release of doxorubicin

This paper describes the synthesis and characterization of a pH-responsive drug release system based on cadmium telluride quantum dots capped by fluorescent dye-labeled poly (N-(2-(2-amino-ethylamino)-ethyl methacrylamide) metal-chelating polymer. Fluorescent dye is a model for an active molecule which shows our polymer can be attached to bioactive agents such as antibody or a functional drug. Furthermore, it indicates the success of ligand exchange between anchor groups on polymer surface and QDs. The polymer was synthesized using reversible addition fragmentation chain transfer polymerization, resulting in polymers with controllable molecular weights and low polydispersities. The use of diethylenetriamine allows obtaining hydrophilic polymers with amine functional groups available for conjugation with QDs. Furthermore, the amine moieties of the polymer guarantee the pH sensitivity of the system. The obtained nano carrier was monodispersed with very small diameter around 4.5 nm which confirmed by TEM. The obtained nanoparticles show low cytotoxicity in cell viability studies. The release rates of doxorubicin (DOX) anticancer drug conjugates were studied at pH 5.4 and pH 7.4. The release behavior for DOX in pH 7.4 (pH of normal cells) is <1%, while it is high at pH 5.4 (pH of cancer cells).