Lanthanide-Loaded Nanoparticles as Potential Fluorescent and Mass Probes for High-Content Protein Analysis.

Multiparametric and high-content protein analysis of single cells or tissues cannot be accomplished with the currently available flow cytometry or imaging techniques utilizing fluorophore-labelled antibodies, because the number of spectrally resolvable fluorochromes is limited. In contrast, mass cytometry can resolve more signals by exploiting lanthanide-tagged antibodies; however, only about 100 metal reporters can be attached to an antibody molecule. This makes the sensitivity of lanthanide-tagged antibodies substantially lower than fluorescent reporters. A new probe that can carry more lanthanide molecules per antibody is a desirable way to enhance the sensitivity needed for the detection of protein with low cellular abundance. Herein, we report on the development of new probes utilizing mesoporous silica nanoparticles (MSNPs) with hydroxyl, amine, or phosphonate functional groups. The phosphonated MSNPs proved to be best at loading lanthanides for up to 1.4 × 106 molecules per particle, and could be loaded with various lanthanide elements (Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, and Lu) at relatively similar molar extents. The modified MSNPs can also load a fluorescent dye, allowing bimodal mass and fluorescence-based detection. We achieved specificity of antibody-conjugated nanoparticles (at 1.4 × 10³ antibodies per nanoparticle) for targeting proteins on the cell surface. The new materials can potentially be used as mass cytometry probes and provide a method for simultaneous monitoring of a large host of factors comprising the tumor microenvironment (e.g., extracellular matrix, cancer cells, and immune cells). These novel probes may also benefit personalized medicine by allowing for high-throughput analysis of multiple proteins in the same specimen.

Removal of a gadolinium based contrast agent by a novel sorbent hemoperfusion in a chronic kidney disease (CKD) rodent model.

Gadolinium based contrast agents (GBCAs) have been linked to toxicity in patients, regardless of having impaired or normal renal function. Currently, no therapy is considered highly effective for removing gadolinium (Gd) from the body. We propose a new strategy to reduce blood Gd content that facilitates whole body removal of Gd using a hemoperfusion system consisting of a cartridge of porous silica beads (Davisil®) functionalized with 1,2-hydroxypyridinone (1,2-HOPO). Herein, we report optimization of the hemoperfusion system using an ex vivo blood and an in vivo rat model of chronic kidney disease (CKD). In our ex vivo system, 1,2-HOPO-Davisil outperformed Gambro activated charcoal (AC), which is commonly used in clinical hemoperfusion of aqueous toxins, in terms of Gd capture capacity and rate. In the CKD rat model, the 1,2-HOPO-Davisil hemoperfusion system removed Gd by 3.4 times over the Gambro AC system. 1,2-HOPO-Davisil did not change complete blood counts and common blood biochemistry. Thus, this strategy has great potential for clinical translation to manage GBCAs after magnetic resonance imaging (MRI), before Gd can deposit in the body and cause long-term toxicity. Although gadodiamide was used as a proof of concept model for GBCAs in this study, 1,2-HOPO functionalized mesoporous silica could also capture dissociated Gd and other GBCAs.

Selective capture of radionuclides (U, Pu, Th, Am and Co) using functional nanoporous sorbents.

This work evaluated sorbent materials created from nanoporous silica self-assembled with monolayer (SAMMS) of hydroxypyridinone derivatives (1,2-HOPO, 3,2-HOPO, 3,4-HOPO), acetamide phosphonate (Ac-Phos), glycine derivatives (IDAA, DE4A, ED3A), and thiol (SH) for capturing of actinides and transition metal cobalt. In filtered seawater doped with competing metals (Cr, Mn, Fe, Co, Cu, Zn, Se, Mo) at levels encountered in environmental or physiological samples, 3,4-HOPO-SAMMS was best at capturing uranium (U(VI)) from pH 2-8, Ac-Phos and 1,2-HOPO-SAMMS sorbents were best at pH < 2. 3,4-HOPO-SAMMS effectively captured thorium (Th(IV)) and plutonium (239Pu(IV)) from pH 2-8, and americium (241Am(III)) from pH 5-8. Capturing cobalt (Co(II)) from filtered river water doped with competing metals (Cu, As, Ag, Cd, Hg, Tl, and Pb) was most effective from pH 5-8 with binding affinity ranged from IDAA > DE4A > ED3A > Ac-Phos > SH on SAMMS. Iminodiacetic acid (IDAA)-SAMMS was also outstanding at capturing Co(II) in ground and seawater. Within 5 min, over 99% of U(VI) and Co(II) in seawater was captured by 3,4-HOPO-SAMMS and IDAA-SAMMS, respectively. These nanoporous materials outperformed the commercially available cation sorbents in binding affinity and adsorption rate. They have great potential for water treatment and recovery of actinides and cobalt from complex matrices.

Lyophilization and stability of antibody-conjugated mesoporous silica nanoparticle with cationic polymer and PEG for siRNA delivery.

INTRODUCTION: Long-term stability of therapeutic candidates is necessary toward their clinical applications. For most nanoparticle systems formulated in aqueous solutions, lyophilization or freeze-drying is a common method to ensure long-term stability. While lyophilization of lipid, polymeric, or inorganic nanoparticles have been studied, little has been reported on lyophilization and stability of hybrid nanoparticle systems, consisting of polymers, inorganic particles, and antibody. Lyophilization of complex nanoparticle systems can be challenging with respect to preserving physicochemical properties and the biological activities of the materials. We recently reported an effective small-interfering RNA (siRNA) nanoparticle carrier consisting of 50-nm mesoporous silica nanoparticles decorated with a copolymer of polyethylenimine and polyethyleneglycol, and antibody. MATERIALS AND METHODS: Toward future personalized medicine, the nanoparticle carriers were lyophilized alone and loaded with siRNA upon reconstitution by a few minutes of simple mixing in phosphate-buffered saline. Herein, we optimize the lyophilization of the nanoparticles in terms of buffers, lyoprotectants, reconstitution, and time and temperature of freezing and drying steps, and monitor the physical and chemical properties (reconstitution, hydrodynamic size, charge, and siRNA loading) and biological activities (gene silencing, cancer cell killing) of the materials after storing at various temperatures and times. RESULTS: The material was best formulated in Tris-HCl buffer with 5% w/w trehalose. Freezing step was performed at -55°C for 3 h, followed by a primary drying step at -40°C (100 µBar) for 24 h and a secondary drying step at 20°C (20 µBar) for 12 h. The lyophilized material can be stored stably for 2 months at 4°C and at least 6 months at -20°C. CONCLUSION: We successfully developed the lyophilization process that should be applicable to other similar nanoparticle systems consisting of inorganic nanoparticle cores modified with cationic polymers, PEG, and antibodies.

Targeted Treatment of Metastatic Breast Cancer by PLK1 siRNA Delivered by an Antioxidant Nanoparticle Platform.

Metastatic breast cancer is developed in about 20% to 30% of newly diagnosed patients with early-stage breast cancer despite treatments. Herein, we report a novel nanoparticle platform with intrinsic antimetastatic properties for the targeted delivery of Polo-like kinase 1 siRNA (siPLK1). We first evaluated it in a triple-negative breast cancer (TNBC) model, which shows high metastatic potential. PLK1 was identified as the top therapeutic target for TNBC cells and tumor-initiating cells in a kinome-wide screen. The platform consists of a 50-nm mesoporous silica nanoparticle (MSNP) core coated layer-by-layer with bioreducible cross-linked PEI and PEG polymers, conjugated with an antibody for selective uptake into cancer cells. siRNA is loaded last and fully protected under the PEG layer from blood enzymatic degradation. The material has net neutral charge and low nonspecific cytotoxicity. We have also shown for the first time that the MSNP itself inhibited cancer migration and invasion in TNBC cells owing to its ROS- and NOX4-modulating properties. In vivo, siPLK1 nanoconstructs (six doses of 0.5 mg/kg) knocked down about 80% of human PLK1 mRNA expression in metastatic breast cancer cells residing in mouse lungs and reduced tumor incidence and burden in lungs and other organs of an experimental metastasis mouse model. Long-term treatment significantly delayed the onset of death in mice and improved the overall survival. The platform capable of simultaneously inhibiting the proliferative and metastatic hallmarks of cancer progression is unique and has great therapeutic potential to also target other metastatic cancers beyond TNBC. Mol Cancer Ther; 16(4); 763-72. ©2017 AACR.

Dermal delivery of HSP47 siRNA with NOX4-modulating mesoporous silica-based nanoparticles for treating fibrosis.

Fibrotic diseases such as scleroderma have been linked to increased oxidative stress and upregulation of pro-fibrotic genes. Recent work suggests a role of NADPH oxidase 4 (NOX4) and heat shock protein 47 (HSP47) in inducing excessive collagen synthesis, leading to fibrotic diseases. Herein, we elucidate the relationship between NOX4 and HSP47 in fibrogenesis and propose to modulate them altogether as a new strategy to treat fibrosis. We developed a nanoparticle platform consisting of polyethylenimine (PEI) and polyethylene glycol (PEG) coating on a 50-nm mesoporous silica nanoparticle (MSNP) core. The nanoparticles effectively delivered small interfering RNA (siRNA) targeting HSP47 (siHSP47) in an in vitro model of fibrosis based on TGF-ß stimulated fibroblasts. The MSNP core also imparted an antioxidant property by scavenging reactive oxygen species (ROS) and subsequently reducing NOX4 levels in the in vitro fibrogenesis model. The nanoparticle was far superior to n-acetyl cysteine (NAC) at modulating pro-fibrotic markers. In vivo evaluation was performed in a bleomycin-induced scleroderma mouse model, which shares many similarities to human scleroderma disease. Intradermal administration of siHSP47-nanoparticles effectively reduced HSP47 protein expression in skin to normal level. In addition, the antioxidant MSNP also played a prominent role in reducing the pro-fibrotic markers, NOX4, alpha smooth muscle actin (α-SMA), and collagen type I (COL I), as well as skin thickness of the mice.

Cationic Polymer Modified Mesoporous Silica Nanoparticles for Targeted SiRNA Delivery to HER2+ Breast Cancer.

In vivo delivery of siRNAs designed to inhibit genes important in cancer and other diseases continues to be an important biomedical goal. We now describe a new nanoparticle construct that has been engineered for efficient delivery of siRNA to tumors. The construct is comprised of a 47-nm mesoporous silica nanoparticle (MSNP) core coated with a cross-linked PEI-PEG copolymer, carrying siRNA against the HER2 oncogene, and coupled to the anti-HER2 monoclonal antibody (trastuzumab). The construct has been engineered to increase siRNA blood half-life, enhance tumor-specific cellular uptake, and maximize siRNA knockdown efficacy. The optimized anti-HER2-nanoparticles produced apoptotic death in HER2 positive (HER2+) breast cancer cells grown in vitro, but not in HER2 negative (HER2-) cells. One dose of the siHER2-nanoparticles reduced HER2 protein levels by 60% in trastuzumab-resistant HCC1954 xenografts. Multiple doses administered intravenously over 3 weeks significantly inhibited tumor growth (p < 0.004). The siHER2-nanoparticles have an excellent safety profile in terms of blood compatibility and low cytokine induction, when exposed to human peripheral blood mononuclear cells. The construct can be produced with high batch-to-batch reproducibility and the production methods are suitable for large-scale production. These results suggest that this siHER2-nanoparticle is ready for clinical evaluation.

Novel oral detoxification of mercury, cadmium, and lead with thiol-modified nanoporous silica.

We have developed a thiol-modified nanoporous silica material (SH-SAMMS) as an oral therapy for the prevention and treatment of heavy metal poisoning. SH-SAMMS has been reported to be highly efficient at capturing heavy metals in biological fluids and water. Herein, SH-SAMMS was examined for efficacy and safety in both in vitro and in vivo animal models for the oral detoxification of heavy metals. In simulated gastrointestinal fluids, SH-SAMMS had a very high affinity (Kd) for methyl mercury (MeHg(I)), inorganic mercury (Hg(II)), lead (Pb(II)), and cadmium (Cd(II)) and was superior to other SAMMS with carboxylic acid or phosphonic acid ligands or commercially available metal chelating sorbents. SH-SAMMS also effectively removed Hg from biologically digested fish tissue with no effect on most nutritional minerals found in fish. SH-SAMMS could hold Hg(II) and MeHg(I) tightly inside the nanosize pores, thus preventing bacteria from converting them to more absorbable forms. Rats fed a diet containing MeHg(I), Cd(II), and Pb(II) and SH-SAMMS for 2 weeks had blood Hg levels significantly lower than rats fed the metal-rich diet only. Upon cessation of the metal-rich diet, continued administration of SH-SAMMS for 2 weeks facilitated faster and more extensive clearance of Hg than in animals not continued on oral SH-SAMMS. Rats receiving SH-SAMMS also suffered less weight loss as a result of the metal exposure. Retention of Hg and Cd in major organs was lowest in rats fed with SH-SAMMS throughout the entire four weeks. The reduction of blood Pb by SH-SAMMS was significant. SH-SAMMS was safe to intestinal epithelium model (Caco-2) and common intestinal bacteria (Escherichia coli). Altogether, it has great potential as a new oral drug for the treatment of heavy metal poisoning. This new application is enabled by the installation of tailored interfacial chemistry upon nontoxic nanoporous materials.

Nanoporous sorbent material as an oral phosphate binder and for aqueous phosphate, chromate, and arsenate removal.

Phosphate removal is both biologically and environmentally important. Biologically, hyperphosphatemia is a critical condition in end-stage chronic kidney disease patients. Patients with hyperphosphatemia are treated long-term with oral phosphate binders to prevent phosphate absorption to the body by capturing phosphate in the gastrointestinal (GI) tract followed by fecal excretion. Environmentally, phosphate levels in natural water resources must be regulated according to limits set forth by the US Environmental Protection Agency. By utilizing nanotechnology and ligand design, we developed a new material to overcome limitations of traditional sorbent materials such as low phosphate binding capacity, slow binding kinetics, and negative interference by other anions. A phosphate binder based on iron-ethylenediamine on nanoporous silica (Fe-EDA-SAMMS) has been optimized for substrates and Fe(III) deposition methods. The Fe-EDA-SAMMS material had a 4-fold increase in phosphate binding capacity and a broader operating pH window compared to other reports. The material had a faster phosphate binding rate and was significantly less affected by other anions than Sevelamer HCl, the gold standard oral phosphate binder, and AG® 1-X8, a commercially available anion exchanger. It had less cytotoxicity to Caco-2 cells than lanthanum carbonate, another prescribed oral phosphate binder. The Fe-EDA-SAMMS also had high capacity for arsenate and chromate, two of the most toxic anions in natural water.

Improved deposition and deprotection of silane tethered 3,4 hydroxypyridinone (HOPO) ligands on functionalized nanoporous silica.

An improved synthesis of a 3,4 hydroxypyridinone (HOPO) functionalized mesoporous silica is described. Higher 3,4-HOPO monolayer ligand loadings have been achieved, resulting in better performance. Performance improvements were demonstrated with the capture of U(VI) from human blood, plasma and filtered river water.

Functional sorbents for selective capture of plutonium, americium, uranium, and thorium in blood.

Self-assembled monolayer on mesoporous supports (SAMMS) are hybrid materials created from attachment of organic moieties onto very high surface area mesoporous silica. SAMMS with surface chemistries including three isomers of hydroxypyridinone, diphosphonic acid, acetamide phosphonic acid, glycinyl urea, and diethylenetriamine pentaacetate (DTPA) analog were evaluated for chelation of actinides ((239)Pu, (241)Am, uranium, thorium) from blood. Direct blood decorporation using sorbents does not have the toxicity or renal challenges associated with traditional chelation therapy and may have potential applications for critical exposure cases, reduction of nonspecific dose during actinide radiotherapy, and for sorbent hemoperfusion in renal insufficient patients, whose kidneys clear radionuclides at a very slow rate. Sorption affinity (K(d)), sorption rate, selectivity, and stability of SAMMS were measured in batch contact experiments. An isomer of hydroxypyridinone (3,4-HOPO) on SAMMS demonstrated the highest affinity for all four actinides from blood and plasma and greatly outperformed the DTPA analog on SAMMS and commercial resins. In batch contact, a fifty percent reduction of actinides in blood was achieved within minutes, and there was no evidence of protein fouling or material leaching in blood after 24 h. The engineered form of SAMMS (bead format) was further evaluated in a 100-fold scaled-down hemoperfusion device and showed no blood clotting after 2 h. A 0.2 g quantity of SAMMS could reduce 50 wt.% of 100 ppb uranium in 50 mL of plasma in 18 min and that of 500 dpm mL(-1) in 24 min. 3,4-HOPO-SAMMS has a long shelf-life in air and at room temperature for at least 8 y, indicating its feasibility for stockpiling in preparedness for an emergency. The excellent efficacy and stability of SAMMS materials in complex biological matrices suggest that SAMMS can also be used as orally administered drugs and for wound decontamination. By changing the organic groups of SAMMS, they can be used not only for actinides but also for other radionuclides. By using the mixture of these SAMMS materials, broad spectrum decorporation of radionuclides is very feasible.

Selective capture of cesium and thallium from natural waters and simulated wastes with copper ferrocyanide functionalized mesoporous silica.

Copper(II) ferrocyanide on mesoporous silica (FC-Cu-EDA-SAMMS) has been evaluated against iron(III) hexacyanoferrate(II) (insoluble Prussian Blue) for removing cesium (Cs(+)) and thallium (Tl(+)) from natural waters and simulated acidic and alkaline wastes. From pH 0.1-7.3, FC-Cu-EDA-SAMMS had greater affinities for Cs and Tl and was less affected by the solution pH, competing cations, and matrices. SAMMS also outperformed Prussian Blue in terms of adsorption capacities (e.g., 21.7 versus 2.6 mg Cs/g in acidic waste stimulant (pH 1.1), 28.3 versus 5.8 mg Tl/g in seawater), and rate (e.g., over 95 wt% of Cs was removed from seawater after 2 min with SAMMS, while only 75 wt% was removed with Prussian Blue). SAMMS also had higher stability (e.g., 2.5-13-fold less Fe dissolved from 2 to 24 h of contact time). In addition to environmental applications, SAMMS has great potential to be used as orally administered drug for limiting the absorption of radioactive Cs and toxic Tl in gastrointestinal tract.

Selective removal of copper(II) from natural waters by nanoporous sorbents functionalized with chelating diamines.

Copper has been identified as a pollutant of concern by the U.S. Environmental Protection Agency (EPA) because of its widespread occurrence and toxic impact in the environment. Three nanoporous sorbents containing chelating diamine functionalities were evaluated for Cu(2+) adsorption from natural waters: ethylenediamine functionalized self-assembled monolayers on mesoporous supports (EDA-SAMMS), ethylenediamine functionalized activated carbon (AC-CH(2)-EDA), and 1,10-phenanthroline functionalized mesoporous carbon (Phen-FMC). The pH dependence of Cu(2+) sorption, Cu(2+) sorption capacities, rates, and selectivity of the sorbents were determined and compared with those of commercial sorbents (Chelex-100 ion-exchange resin and Darco KB-B activated carbon). All three chelating diamine sorbents showed excellent Cu(2+) removal (approximately 95-99%) from river water and seawater over the pH range 6.0-8.0. EDA-SAMMS and AC-CH(2)-EDA demonstrated rapid Cu(2+) sorption kinetics (minutes) and good sorption capacities (26 and 17 mg Cu/g sorbent, respectively) in seawater, whereas Phen-FMC had excellent selectivity for Cu(2+) over other metal ions (e.g., Ca(2+), Fe(2+), Ni(2+), and Zn(2+)) and was able to achieve Cu below the EPA recommended levels for river and sea waters.

Phosphate removal by anion binding on functionalized nanoporous sorbents.

Phosphate was captured from aqueous solutions by cationic metal-EDA complexes anchored inside mesoporous silica MCM-41 supports (Cu(II)-EDA-SAMMS and Fe(III)-EDA-SAMMS). Fe-EDA-SAMMS was more effective at capturing phosphate than the Cu-EDA-SAMMS and was further studied for matrix effects (e.g., pH, ionic strength, and competing anions) and sorption performance (e.g., capacity and rate). The adsorption of phosphate was highly pH dependent; it increased with increasing pH from 1.0 to 6.5, and decreased above pH 6.5. The adsorption was affected by high ionic strength (0.1 M of NaCl). In the presence of 1000-fold molar excess of chloride and nitrate anions, phosphate removal by Fe-EDA-SAMMS was not affected. Slight, moderate and large impacts were seen with bicarbonate, sulfate, and citrate anions, respectively. The phosphate adsorption data on Fe-EDA-SAMMS agreed well with the Langmuir model with the estimated maximum capacity of 43.3 mg/g. The material displayed rapid sorption rate (99% of phosphate removal within 1 min) and lowering the phosphate content to approximately 10 microg/L of phosphorus, which is lower than the EPA's established freshwater contaminant level for phosphorus (20 microg/L).

New functional materials for heavy metal sorption: "supramolecular" attachment of thiols to mesoporous silica substrates.

A new class of sorbent material, which exhibits exceptional metal capture from contaminated natural water, features aromatic thiol ligands reversibly bound to functionalized mesoporous silica through non-covalent interactions and have the potential of being regenerable.

Direct detection of Pb in urine and Cd, Pb, Cu, and Ag in natural waters using electrochemical sensors immobilized with DMSA functionalized magnetic nanoparticles.

Urine is universally recognized as one of the best non-invasive matrices for biomonitoring exposure to a broad range of xenobiotics, including toxic metals. Detection of metal ions in urine has been problematic due to the protein competition and electrode fouling. For direct, simple, and field-deployable monitoring of urinary Pb, electrochemical sensors employing superparamagnetic iron oxide (Fe3O4) nanoparticles with a surface functionalization of dimercaptosuccinic acid (DMSA) has been developed. The metal detection involves rapid collection of dispersed metal-bound nanoparticles from a sample solution at a magnetic or electromagnetic electrode, followed by the stripping voltammetry of the metal in acidic medium. The sensors were evaluated as a function of solution pH, the binding affinity of Pb to DMSA-Fe3O4, the ratio of nanoparticles per sample volume, preconcentration time, and Pb concentrations. The effect of binding competitions between the DMSA-Fe3O4 and urine constituents for Pb on the sensor responses was studied. After 90 s of preconcentration in samples containing 25 vol.% of rat urine and 0.1 g L(-1) of DMSA-Fe3O4, the sensor could detect background level of Pb (0.5 ppb) and yielded linear responses from 0 to 50 ppb of Pb, excellent reproducibility (%RSD of 5.3 for seven measurements of 30 ppb Pb), and Pb concentrations comparable to those measured by ICP-MS. The sensor could also simultaneously detect background levels (<1 ppb) of Cd, Pb, Cu, and Ag in river and seawater.

Removal of heavy metals from aqueous systems with thiol functionalized superparamagnetic nanoparticles.

We have shown that superparamagnetic iron oxide (Fe3O4) nanoparticles with a surface functionalization of dimercaptosuccinic acid (DMSA) are an effective sorbent material for toxic soft metals such as Hg, Ag, Pb, Cd, and Tl, which effectively bind to the DMSA ligands and for As, which binds to the iron oxide lattices. The nanoparticles are highly dispersible and stable in solutions, have a large surface area (114 m2/g), and have a high functional group content (1.8 mmol thiols/g). They are attracted to a magnetic field and can be separated from solution within a minute with a 1.2 T magnet. The chemical affinity, capacity, kinetics, and stability of the magnetic nanoparticles were compared to those of conventional resin based sorbents (GT-73), activated carbon, and nanoporous silica (SAMMS) of similar surface chemistries in river water, groundwater, seawater, and human blood and plasma. DMSA-Fe3O4 had a capacity of 227 mg of Hg/g, a 30-fold larger value than GT-73. The nanoparticles removed 99 wt% of 1 mg/L Pb within a minute, while it took over 10 and 120 min for Chelex-100 and GT-73 to remove 96% of Pb.