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The high prevalence of breast cancer is a global health concern, but there are no safe or effective treatments for it at its advanced stages. These facts urge the development of novel treatment strategies. Annexin A5 (ANXA5) is a natural human protein that binds with high specificity to phosphatidylserine, a phospholipid tightly maintained in the inner leaflet of the cell membrane on most healthy cells but externalized in tumor cells and the tumor vasculature. Here, we have developed a targeted photosensitizer for photothermal therapy (PTT) of solid tumors through the functionalization of single walled carbon nanotubes (SWCNTs) to ANXA5—the SWCNT-ANXA5 conjugate. The ablation of tumors through the SWCNT-ANXA5-mediated PTT synergizes with checkpoint inhibition, creating a systemic anti-cancer immune response. In vitro ablation of cells incubated with the conjugate promoted cell death in a dose-dependent and targeted manner. This treatment strategy was tested in vivo with the orthotopic EMT6 breast tumor model in female balb/cJ mice. Enhanced therapeutic effects were achieved by using intratumoral injection of the conjugate and treating tumors at a lower PTT temperature (45oC). When combined with checkpoint inhibition of anti-PD-1, SWCNT-ANXA5-mediated PTT increased survival and 80% of the mice survived for 100 days. Evidence of immune system activation by flow cytometry of splenic cells strengthens the hypothesis of an abscopal effect as a mechanism of prolonged survival.

This file contains includes 9 figures of supplementary data (S1-S9) and a description of live cell microscopy and the assessment of synergism for the combination therapies.

Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with Annexin A1 or Annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart nonnative methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of nontoxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of nonnative proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple-negative murine 4T1 mammary tumors in immunocompetent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in the occurrence of negative side effects or the elicitation of neutralizing antibodies. On the basis of the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an mTOR inhibitor, reduces the prosurvival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. IHC revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia-inducible factor 1 α subunit, HIF1A) in the enzyme prodrug–treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. Mol Cancer Ther; 16(9); 1855–65. ©2017 AACR.

Copper(II) is known to bind in the influenza virus His37 cluster in the homotetrameric M2 proton channel and block the proton current needed for uncoating. Copper complexes based on iminodiacetate also block the M2 proton channel and show reduced cytotoxicity and zebrafish-embryo toxicity. In voltage-clamp oocyte studies using the ubiquitous amantadine-insensitive M2 S31N variant, the current block showed fast and slow phases, in contrast to the single phase found for amantadine block of wild-type M2. Here, we evaluate the mechanism of block by copper adamantyl iminodiacitate and copper cyclooctyl iminodiacitate complexes and address whether the complexes can coordinate with one or more of the His37 imidazoles. The current traces were fitted to parametrized master equations. The energetics of binding and the rate constants suggest that the first step is copper complex binding within the channel, and the slow step in the current block is the formation of a Cu-histidine coordination complex. Solution-phase isothermal titration calorimetry and density functional theory (DFT) calculations indicate that imidazole binds to the copper complexes. Structural optimization using DFT reveals that the complexes fit inside the channel and project the Cu(II) toward the His37 cluster, allowing one imidazole to form a coordination complex with Cu(II). Electrophysiology and DFT studies also show that the complexes block the G34E amantadine-resistant mutant despite some crowding in the binding site by the glutamates.

Due to their non-toxicity and their ability to be functionalized, magnetite (Fe3O4) nanoparticles (NPs) are good candidates for a variety of biomedical applications. To better implement their applications, it is crucial to well understand the basic structural and magnetic properties of the NPs in correlation with their synthesis method. Here, we show interesting properties of Fe3O4 NPs of various sizes ranging from 5 to 100 nm and the dependence of these properties on particle size and preparation method. One synthetic method based on heating Fe(acac)3 with oleic acid consistently gives 5 ± 1 nm NPs. A second method using the thermal decomposition of Fe(oleate)3 in oleic acid led to larger NPs, greater than 8 nm in size. Increasing the amount of oleic acid caused the average NP size to slightly increase from 8 to 10 nm. Increasing both the reaction temperature and the reaction time caused the NP size to drastically increase from 10 to 100 nm. Powder X-ray diffraction and electron-microscopy imaging show a pure single crystalline Fe3O4 phase for all NPs smaller than 50 nm and spherical in shape. When the NPs get larger than 50 nm, they notably tend to form faceted, FeO core–Fe3O4 shell structures. Magnetometry data collected in various field-cooling conditions show a pure superparamagnetic (SPM) behavior for all NPs smaller than 20 nm. The observed blocking temperature, $T_{B}$ , gradually increases with NP size from about 25–150 K. In addition, the Verwey transition is observed with the emergence of a strong narrow peak at 125 K in the magnetization curves when larger NPs are present. Our data confirm the vanishing of the Verwey transition in smaller NPs. Magnetization loops indicate that the saturating field drastically decreases with NP size. While larger NPs show some coercivity ( $H_{c}$ ) up to 30 mT at 400 K, NPs smaller than 20 nm show no coercivity ( $H_{c} = 0$ ), confirming their pure SPM behavior at high temperature. Upon cooling below $T_{B}$ , some of the SPM NPs gradually show some coercivity, with $H_{c}$ reaching 45 mT at 5 K for the 10 nm NPs, indicating emergent interparticle couplings in the blocked state.

Resorcinarene-based deep cavitands including glutamic acid methyl resorcinarene (GMA) and glutamic acid undecyl resorcinarene (GUA) have been synthesised and their amine binding properties analysed. The cavitands are designed with hydrogen bonding groups on their upper rim (glutamic acids) and hydrophobic groups, on their lower rims. Along with the aromatic groups of the resorcinarene, these cavitands have many sites for guest binding. The cavitands form kite-like structures in dimethylsulfoxide, acetone, and water. The binding of GMA with amines, including guanidine containing drug compounds, was studied in DMSO by UV-vis titration and compared to that of phthalyl glutamic acid. The obtained binding constants for GMA to amines (K values) were in the range of 12–136 × 103 M−1 for benzyl amines and 4.3–153 × 103 M−1 for guanidine compounds. GMA shows a binding preference for more basic and less sterically encumbered amines and slightly stronger binding to S-enantiomers.

Identifying a universal biomarker for cancer treatment remains a major challenge in cancer therapy. Extracellular exposure of phosphatidylserine (PS) is tightly regulated and is an "eat me" signal for phagocytosis in healthy cells. Although cancer cells and vasculature express high levels of externalized PS, they do not undergo apoptosis, making them a promising biomarker for cancer treatment. Annexin A5 (ANXA5) is the native binding partner of PS and can actively target and deliver chemotherapies to the tumor microenvironment (TME) via PS expression. ANXA5 acts as a bridge between the innate and adaptive immune systems and contributes to an immunostimulatory profile in the TME. ANXA5-enzyme prodrug therapies allow for systemic delivery of prodrugs and targeted killing at the tumor site. ANXA5-carbon nanotube conjugates have been used to physically ablate tumors via photothermal therapy. This review aims to explore the expression of PS in cancer cells and how ANXA5 has been used as a chemotherapeutic and targeting agent for cancer.

The magnetic properties of ${\mathrm{Fe}}_{3}{\mathrm{O}}_{4}$ nanoparticle assemblies have been investigated in detail through a combination of vibrating sample magnetometry (VSM) and muon spin relaxation ($\ensuremath{\mu}\mathrm{S}\mathrm{R}$) techniques. Two samples with average particle sizes of 5 and 20 nm, respectively, were studied. For both samples, the VSM and $\ensuremath{\mu}\mathrm{S}\mathrm{R}$ results exhibit clear signatures of superparamagnetism at high temperature and magnetic blocking at low temperature. The $\ensuremath{\mu}\mathrm{S}\mathrm{R}$ data demonstrate that the transition from the superparamagnetic to the blocked state occurs gradually throughout the sample volume over an extended temperature range due to the finite particle size distribution of each nanoparticle batch. The transition occurs between approximately 3 and 45 K for the 5-nm nanoparticles and 150 and 300 K for the 20-nm nanoparticles. The VSM and $\ensuremath{\mu}\mathrm{S}\mathrm{R}$ data are further analyzed to yield estimates of microscopic magnetic parameters including the nanoparticle spin-flip activation energy ${E}_{A}$, magnetic anisotropy $K$, and intrinsic nanoparticle spin reversal attempt time ${\ensuremath{\tau}}_{0}$. These results highlight the complementary information about magnetic nanoparticles that can be obtained by bulk magnetic probes such as magnetometry and local magnetic probes such as $\ensuremath{\mu}\mathrm{S}\mathrm{R}$.

The greatest contributors to cancer mortality are metastasis and the consequences of its treatment. Here, we present a novel treatment of metastatic breast cancer that combines photothermal therapy with targeted single-walled carbon nanotubes (SWCNTs) and immunostimulation with a checkpoint inhibitor. We find that the selective near-infrared photothermal ablation of primary orthotopic EMT6 breast tumors in syngeneic BALB/cJ mice using an annexin A5 (ANXA5) functionalized SWCNT bioconjugate synergistically enhances an anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4)-dependent abscopal response, resulting in an increased survival (55%) at 100 days after tumor inoculation. In comparison, there was no survival at 100 days for either photothermal therapy by itself or immunostimulation by itself. Prior to photothermal therapy, the SWCNT-ANXA5 bioconjugate was administered systemically at a relatively low dose of 1.2 mg/kg, where it then accumulated in tumor vasculature via ANXA5-dependent binding. During photothermal therapy, the average maximum temperature in the tumor reached 54 °C (duration 175 s). The mechanism of prolonged survival resulting from combinatorial photothermal ablation and immune stimulation was evaluated by flow cytometric quantification of splenic antitumoral immune effector cells and serum cytokine quantification.

Copper(II) is known to bind in the influenza virus His37 cluster in the homotetrameric M2 proton channel and block the proton current needed for uncoating. Copper complexes based on iminodiacetate also block the M2 proton channel and show reduced cytotoxicity and zebrafish-embryo toxicity. In voltage-clamp oocyte studies using the ubiquitous amantadine-insensitive M2 S31N variant, the current block showed fast and slow phases in contrast to the single phase found for amantadine block of WT M2. Here we evaluate the mechanism of block by copper adamantyl iminodiacitate (Cu(AMT-IDA)) and copper cyclooctyl iminodiacitate (Cu(CO-IDA)) complexes and address whether the complexes can covalently bind to one or more of the His37 imidazoles. The current traces were fitted to parametrized master equations. The energetics of binding and the rate constants suggest that the first step is copper-complex binding within the channel and the slow step in the current block is the covalent bond formation between copper complex and histidine. Isothermal titration calorimetry (ITC) indicates that a single imidazole binds strongly to the copper complexes. Structural optimization using density functional theory (DFT) reveals that the complexes fit inside the channel and project the Cu(II) towards the His37 cluster allowing one imidazole to form a covalent bond with the Cu(II). Electrophysiology and DFT studies also show that the complexes block the G34E amantadine-resistant mutant in spite of some crowding in the binding site by the glutamates.

Technetium poses an environmental hazard because of its radioactivity and long half-life. It exists in the form of pertechnetate in the environment and can be modeled by the nonradioactive ion perrhenate, since pertechnetate and perrhenate have the same geometry and similar chemical properties. In this research, a new zinc cyclen resorcinarene cavitand (ZCR) column was used in ion chromatography (IC) to efficiently separate perrhenate. Ion chromatography has the advantage of requiring almost no sample preparation for water samples. The ZCR column demonstrated the ability to separate anions: fluoride, chloride, nitrate, sulfate, phosphate, perchlorate, and perrhenate by gradient 2–60 mM NaOH. Unlike other columns, the new column material was selective in retaining perrhenate. The ZCR column also gave a linear range from 2.0 to 1000 mg L−1 for perrhenate with R2 > 0.997. There was a logarithmic relationship between the concentration of perrhenate and its retention time. Excellent perrhenate recovery was achieved on the ZCR column when river water was spiked with perrhenate and perrhenate was preconcentrated. The efficient separations of perrhenate by the ZCR column will potentially assist in pertechnetate separations.

This study validates bacterial anionic phospholipids (APs) as a putative molecular target in a novel antibiotic treatment against the Gram-positive bacterium Listeria monocytogenes and the Gram-negative bacterium Escherichia coli. Bacterial AP expression was targeted with its associated protein-ligand partner, annexin A5 (ANXA5). This protein was functionalised with the covalent addition of the antibiotic ampicillin (AMP) and separately with the antibiotic moxifloxacin (MOX). Functionalised ANXA5 serves as a delivery vehicle, directing the antibiotic to bacterial AP expression. The results presented here suggest that this ANXA5-AMP bioconjugate participates in a positive feedback loop where APs, the target of the delivery vehicle ANXA5, are upregulated by the chemotherapeutic payload of the bioconjugate. Importantly, the ANXA5 delivery vehicle is non-toxic to bacterial cells by itself and neither is the ANXA5–antibiotic bioconjugate toxic to human vascular endothelial cells. As measured by the IC50, conjugation to ANXA5 resulted in increasing the antibiotic activity of AMP against L. monocytogenes and E. coli by more than 4 and 3 orders of magnitude, respectively, compared with free AMP, whilst the activity of MOX against L. monocytogenes is increased by 4 orders of magnitude. Given the conservation of AP expression in pathologies such as oncogenesis and other bacterial/viral/parasitic infections, we hypothesise that a therapeutic modality targeting AP expression may be a viable chemotherapeutic strategy in many infectious diseases.

A new sensor for Hg2+ and cysteine detection has been synthesized and tested. The sensor based on cinamaldehyde and pyrimidine absorbs visible light when exposed to Hg2+ and becomes colored. The sensor only changes color due to Hg2+ and does not change when exposed to some other metal ions. Calculations show the change in color is due to lowering of the HOMO-LUMO transition. Titrations show the sensor-Hg2+ complex is composed of two sensor molecules per Hg2+. The limit of detection for Hg2+ is 0.39 μM. The sensor-Hg2+ complex selectively responds to cysteine (Cys), but does not show a color change due to other amino acids. The limit of detection of Cys by the sensor-Hg2+ complex is 0.10 μM.

Hypochlorite is used as a water disinfectant and it is also produced by biological organisms. Its detection and quantification is important and could lead to its mechanism of reactivity in cells. We have synthesized a new fluorescence sensor for hypochlorite based on bithiophene and furan-carbohydrazide. The sensor shows increased fluorescence as a function of hypochlorite and is selective for hypochlorite. Fluorescence enhancement due to hypochlorite is observed when the sensor is used in aqueous solutions at neutral pH values. Using the sensor, the detection limit for hypochlorite is 4.2 µM, making the sensor practical to determine hypochlorite in water. Applying the sensor to aide in the detection of hypochlorite in zebrafish, showed localization of ClO−/HClO in the air bladders and eyes of zebrafish.

New M2 blockers effective against the ubiquitous amantadine-resistant S31N M2 mutation in influenza A are needed. Six copper complexes, 2, 4, 6, 8, 9, and 10, were synthesized and found to block both wild type and S31N M2. Free Cu2+ also blocks M2 S31N but not S31N/H37A. The copper complexes do not block M2 H37A (either S31 or S31N). The complexes were effective against three influenza A strains in cell-culture assays, but less toxic to cells than CuCl2. For example 4, Cu(cyclooctylamineiminodiacetate), which was stable at pH > 4 in the buffers used, had an EC50 against A/Calif/07/2009 H1N1 of 0.7 ± 0.1 μM with a CC50 of 147 μM (therapeutic index, averaged over three strains, 67.8). In contrast, CuCl2 had an EC50 of 3.8 ± 0.9 μM and CC50 of 19 μM. Because M2 H37 is highly conserved, these complexes show promise for further testing as drugs against all strains of influenza A.

Formation of crystalline hexagonal ZnO prisms from a sol–gel method is presented. The method requires zinc acetate, water, and diethanolamine to create a zinc hydroxide/zinc hydroxide acetate gel, which in the presence of water and upon heating forms hexagonal prisms. Ethanolamines were found to be critical for gel and prism formation, while other bases such NaOH and triethylamine produce nanoparticles. Scanning electron microscope images showed hexagonal prisms with uniform size (approx. 0.5 × 2 µm) and very distinct edges. Transmission electron microscope and electron diffraction images showed that the prisms were highly crystalline with rough surfaces. Transmission electron microscope also showed a change from randomly oriented particles in the gel to ordered crystals after heating. Using an X-ray diffraction high-temperature chamber to characterize the gel, it was determined that it formed ZnO at 100 °C. Along with heating, acetate and water were found to be necessary for prism formation. In a potential application, the prisms efficiently acted as a photocatalyst in the degradation of organic dye.

Since their discovery in the 1990s, carbon nanotubes have been intensively studied in several different fields, including recently for the treatment of cancer. A property of carbon nanotubes that has been used extensively in cancer treatment studies is their strong absorbance of light in the near-infrared range (700–1400 nm). This strong absorbance of light leads to heating of the nanotubes and destruction of cancer cells that have taken up or are in the vicinity of nanotubes. Photothermal therapy studies for cancer treatment both in vitro and in vivo will be discussed, including the functionalization of the nanotubes for targeting and also the in vivo coadministration of other agents such as antibodies to stimulate the immune system and thereby increase the therapeutic response. For the work in vivo, research will be discussed about the biodistribution of carbon nanotubes after being injected and their elimination and/or degradation, including toxicology evaluations.

"Organization and Strategy, Roger G. Harrison Analytical Considerations in the Development of Protein Purification Processes, Vincent Anicetti and William S. Hancock Cell Breakage, Cady R. Engler Crossflow Membrane Filtration, Zhi-Guo Su and Clark K. Cotton Liquid-Liquid Extraction, Dennis J. Kubek Differential Precipitation of Proteins: Science and Technology, Fred Rothstein Conventional Chromatography, John M. Simpson Biospecific Affinity Chromatography, Nikos K. Harakas Freeze Drying: A Practical Overview, Larry A. Gatlin and Steven L. Nail "

Following our call to join in the discussion over the suitability of implementing a reporting checklist for bio–nano papers, the community responds.

Objectives The bleak prognosis associated with pancreatic cancer (PDAC) drives the need for the development of novel treatment methodologies. Here, we evaluate the applicability of 3 enzyme prodrug therapies for PDAC, which are simultaneously targeted to the tumor, tumor vasculature, and metastases via annexin V. In these therapies, annexin V is fused to an enzyme, creating a fusion protein that converts nontoxic drug precursors, prodrugs, into anticancer compounds while bound to the tumor, therefore mitigating the risk of side effects. Methods The binding strength of fusion proteins to the human PDAC cell lines Panc-1 and Capan-1 was measured via streptavidin-horseradish peroxidase binding to biotinylated fusion proteins. Cytotoxic efficacy was evaluated by treatment with saturating concentrations of fusion protein followed by varying concentrations of the corresponding prodrug plus docetaxel. Results All fusion proteins exhibited strong binding to PDAC cells, with dissociation constants between 0.02 and 1.15 nM. Cytotoxic efficacy was determined to be very good for 2 of the systems, both of which achieved complete cell death on at least 1 cell line at physiologically attainable prodrug concentrations. Conclusions Strong binding of fusion proteins to PDAC cells and effective cytotoxicity demonstrate the potential applicability of enzyme prodrug therapy to the treatment of PDAC.

Monitoring Zn2+ levels in biological environments with fluorescent sensors is important. This paper gives the synthesis and properties of a new Zn2+ sensor based on quinoline and pyridylaminophenol. The sensor is selective for Zn2+ and remains fluorescent when bound to Zn2 even in the presence of other metal ions. Along with fluorescing when bound to Zn2+, the sensor becomes colored when Cu2+ or Co2+ is added to it. These two metal ions result in the sensor becoming yellow. The crystal structure of the Cu–sensor complex shows that all of the sensor's nitrogens are bound to the metal ion. In studies with living cells, the fluorescence intensity of the sensor correlates to the concentration of Zn2+.

The extent of reduction of unsupported iron-based high-temperature water-gas shift catalysts with small (

Objectives Use of electronic cigarettes has dramatically increased in the United States since 2010, with a forecasted growth of 37% between 2014 and 2019. There is little research on e-liquid nicotine concentration from domestic manufacturers. However, limited research outside of the United States found wide inconsistencies between the labeled concentration of nicotine in e-liquids and the actual nicotine concentration. Methods The 7 most popular online manufacturers or distributors were identified. E-liquid samples of the 5 most popular flavors from each manufacturer were purchased in nicotine concentrations of 0 and 18 mg/mL. Of the samples purchased (n = 70), all were labeled as produced in the United States of America. The researchers anonymized the samples before sending them to an independent university laboratory for testing. Results The 35 e-liquid samples labeled 18 mg/mL nicotine measured between 11.6 and 27.4 mg/mL (M = 18.7, SD = 3.3) nicotine. The labeled 18 mg/mL samples measured as little as 35% less nicotine and as much as 52% greater nicotine. In the 35 samples labeled 0 mg/mL, nicotine was detected (>0.01 mg/mL) in 91.4% of the samples (range 0-23.9 mg/mL; M = 2.9, SD = 7.2). Six samples from 2 manufacturers labeled as 0 mg/mL were found to contain nicotine in amounts ranging from 5.7 to 23.9 mg/mL. Conclusion This study demonstrates the nicotine labeling inaccuracies present in current e-liquid solutions produced in the United States. Incorrect labeling poses a significant risk to consumers and supports the recent regulation changes enacted by the US Food and Drug Administration. Additional routine testing of nicotine concentrations should be conducted to evaluate the effectiveness of the regulations on future e-liquid production.

Approximately 75% of ovarian cancer is diagnosed once metastasis to the peritoneal cavity has occurred. A large proportion of patients eventually develop platinum-resistive tumors, which are considered terminal. In order to provide an alternative a novel fusion protein, mCTH-ANXA5, has been developed for the treatment of recurrent, metastatic ovarian cancer. The fusion protein combines annexin V (ANXA5), an ovarian tumor and tumor vasculature targeting protein, with mutated cystathionine gamma-lyase (mCTH), an enzyme that converts selenomethionine (SeMet) into toxic methylselenol, which generates reactive oxygen species and eventual tumor cell death. In order to further enhance the therapeutic efficacy, anti-CD73 and anti-OX40 immunostimulants were combined with mCTH-ANXA5, resulting in an increase of survival by 100% from 12 to 24 days post-therapy and decrease tumor burden in mice with orthotopic metastatic ovarian cancer. Further evaluation of the combination therapy revealed a strong antibody-mediated immune response, and an increased infiltration of cytotoxic T-cells along with a decrease in tumor promoting immune cells. This study demonstrates the efficacy of a synergistic, multi-drug system by attacking the tumor as well as enlisting the body's own defense system to treat the patient.

A series of novel chloro-complexes of Mn2+, Co2+, Ni2+ and Fe3+ with 2,4,6-tris-(2-pyridyl)-1,3,5-triazine (L1) and thiourea (L2) with the formulae, [Mn(L1)Cl2(EtOH)]L2 (1), [Co(L1)Cl2(H2O)]L2.1/2H2O (2), [Ni(L1)Cl2(H2O)]L2.1/2H2O (3) and [Fe(L1)Cl3]L2 (4) were investigated utilizing convenient methods. Conversely, similar reaction resulted in complex with structure [Mn(L1)(OAc)(H2O)Cl]·H2O (5) in presence of sodium acetate. Pentagonal-bipyramidal geometry around Mn2+ ion is suggested. The solid state ESR spectra of Mn2+, Co2+ and Ni2+ complexes parameters show g|| > g┴ > 2.0023 indicating that the ground state is dx2-y2. Electronic spectra of two Mn2+ and the other complexes and the values of magnetic moments suggest octahedral geometry for complexes while pentagonal-pyramidal for [Mn(L1)Cl2(EtOH)]L2. Biological activity of complexes was tested versus alpathogenic bacteria (E. Coli, S. Aureus and C. Albicans). Further, the cytotoxic activity was screened for in vitro against (HeLa), (MCF-7), (HePG2) and (HeP-2). The role of ionic radii of the metal ion on the biological activity was investigated.

A zinc sensor based on quinoline and morpholine has been synthesized. The sensor selectively fluoresces in the presence of Zn2+, while not for other metal ions. Absorbance changes in the 350nm region are observed when Zn2+ binds, which binds in a 1:1 ratio. The sensor fluoresces due to Zn2+ above pH values of 6.0 and in the biological important region. The Zn2+-sensor complex has the unique ability to detect both Hg2+ and HS-. The fluorescence of the Zn2+-sensor complex is quenched when it is exposed to aqueous solutions of Hg2+ with sub-micromolar detection levels for Hg2+. The fluorescence of the Zn2+-sensor complex is also quenched by aqueous solutions of hydrosulfide. The sensor was used to detect Zn2+ and Hg2+ in living cells.

Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with Annexin A1 or Annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart nonnative methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of nontoxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of nonnative proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple-negative murine 4T1 mammary tumors in immunocompetent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in the occurrence of negative side effects or the elicitation of neutralizing antibodies. On the basis of the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an mTOR inhibitor, reduces the prosurvival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. IHC revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia-inducible factor 1 α subunit, HIF1A) in the enzyme prodrug–treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. Mol Cancer Ther; 16(9); 1855–65. ©2017 AACR.

INTRODUCTION AND OBJECTIVES: Use of ablative therapy has the potential to treat bladder cancer resistant to BCG and chemotherapy. However, early efforts with photodynamic therapy and intravenous application of the photosensitizer resulted in significant complications. We have combined Annexin V targeting of phosphatidylserine on tumor surface with single-walled carbon nanotubes (SWNTs) to target bladder tumor cells and treat with near infrared (NIR) light for thermal ablation of tumor in the preclinical setting. METHODS: In vitro studies were conducted on mouse bladder cancer (MB49) and human bladder cancer (J82) cell lines. The dissociation constant for AV binding strength was determined. The specific binding results were confirmed with fluorescence microscopy. Quantitative analysis of the number of cell surface bound AVs was conducted for each cell line. Alamar blue preand post-cell viability assays were conducted to determine the cytotoxic effects of NIR and SWNT-AVs alone as well as a combined approach on both bladder cancer lines. In vivo studies were conducted to determine the biodistribution of intravesically delivered SWNT-AVs in MB49 orthotropic models. FT-Raman analysis was conducted to determine SWNT-AV accumulation in harvested organs. An in vivo NIR tolerance test followed by H&E staining was conducted with a 360 radiating fiber to confirm minimized non-specific tissue damage. A therapeutic efficacy study combining SWNT-AVs and NIR was performed. RESULTS: In vitro binding studies confirmed a strong binding affinity of AV to MB49 (Kd 1⁄4 4.14 1.28 nM) and J82 (Kd 1⁄4 0.38 0.20 nM) cells. Subtoxic levels of docetaxel increased the number of bound AVs per MB49 cell but had no effect on the J82 cells. Inducing SWNT-AV heating due to NIR confirmed significant cancer cell death as compared to untreated controls for both cell lines. The in vitro tests provided statistically significant validation for the potential of this targeted ablation therapy. In vivo testing on C57BL-6 mice was conducted to confirm the efficacy of this treatment even further. A biodistribution study followed by FT-Raman analysis verified no non-specific accumulation of SWNT-AVs in various organs. NIR power tolerance tests confirmed that no healthy tissue damage occurred at 50 J/cm2. In vivo data will also be presented for the effect of SWNT-AV and NIR combination therapy on MB49 mouse bladder cancer. CONCLUSIONS: SWNT-AVs have proven to preferentially target bladder cancer cells and in conjunction with NIR cause significant cytotoxicity in vitro. The results of this study show promise for NIR thermally heated SWNT-AVs as a viable therapeutic option for recurrent bladder cancers.

Magnetic nanoparticles are increasingly used in nanotechnologies and biomedical applications, such as drug targeting, MRI, bio-separation. Magnetite (Fe3O4) nanoparticles stand to be effective in these roles due to the non-toxic nature of magnetite and its ease of manufacture. To be more effective in these applications, a greater understanding of the magnetic behavior of a collection of magnetite nanoparticles is needed. This research seeks to discover the local magnetic ordering of ensembles of magnetite nanoparticles occurring under various external fields. To complete this study, we use x-ray resonant magnetic scattering (XRMS). Here we discuss the modeling of the magnetic scattering data using a one-dimensional chain of nanoparticles with a mix of ferromagnetic, anti-ferromagnetic, and random orders. By fitting the model to the experimental data, we extracted information about the magnetic correlations in the nanoparticle assembly.

Influenza A (IAV) M2 proton channel mutations in the primary target site for amantadine cause resistance to its anti-viral drug effect. Though there is still some debate about how this mutation has caused this resistance, it is clear that novel M2 blockers are needed that are effective against the ubiquitous S31N M2 mutation. Divalent copper has previously been shown to have anti-IAV properties using in vitro assays involving block of wild type M2 channels in transfected Xenopus laevis oocytes(1) and binds to the His37 imidazoles according to solid state NMR(2). In this research, the anti-IAV property of divalent copper has been extended to organic complexes of copper. We have synthesized and purified a set of novel copper complexes of derivatized M2 blockers. We have identified a set that are stable in water, purified them of residual free copper through precipitation or crystallization as validated indirectly with inductively coupled plasma mass spec, and tested their stability in typical buffers using UV and Vis absorption spectroscopy. In two-electrode voltage clamp of Xenopus laevis oocytes transfected with A/Udorn/72 M2 S31N, some of these compounds show excellent, poorly reversible block of acid-induced inward currents with low EC50's. In addition, liposome and miniplaque assays, which were previously successful with such compounds(3), are under investigation for the refined set.1. Gandhi et al. 1999. JBC 274:5474-5482.2. Su et al. 2012. JACS 134:8693-8702.3. McGuire et al, 2015. BJ 108:582a.

Trace levels of pharmaceuticals have been detected in surface water and may pose a health risk to humans and other organisms. New chromatographic materials will help identify and quantify these contaminants. We introduce a new ion chromatographic (IC) material designed to separate cationic pharmaceuticals and report its ability to separate a group of guanidine compounds. Guanidine moieties are strongly basic and protonated under acid conditions, and therefore can potentially be separated on the newly designed stationary phase and detected by ion exchange chromatography. The new column packing material is based on glutamic acids bonded to resorcinarene moieties that in turn are bound to divinylbenzene macroporous resin. Detection limits in the range of 5-30 μg L(-1) were achieved using integrated pulsed amperometric detection (IPAD) for guanidine (G), methylguanidine (MG), 1,1-dimethylbiguanide (DMG), agmatine (AGM), guanidinobenzoic acid (GBA) and cimetidine (CIM). Suppressed conductivity (CD) and UV-vis detection resulted in limits of detection similar to IPAD, in the range of 2-66 μg L(-1), but were not able to detect all of the analytes. Three water sources, river, lake, and marsh, were analyzed and despite matrix effects, sensitivity for guanidine compounds was in the 100 μg L(-1) range and apparent recoveries were 80-96%. The peak area precision was 0.01-2.89% for IPAD, CD and UV-vis detection.

Zinc oxide nanomaterials were synthesized with small amounts of magnetic ions to create dilute magnetic semiconductors (DMS), by using a low temperature sol-gel method. Conditions were controlled such that a range of amounts of Co, Ni and Mn were incorporated. The incorporation could be tracked by color changes in the powders to blue for Co, green for Ni and yellow for Mn. XRD measurements showed the ZnO has the wurtzite structure with crystallites 8-12 nm in diameter. Nanoparticles were observed by SEM and TEM and TEM showed that the lattice fringes of different nanoparticles align. Nanoparticle alignment was disrupted when high concentrations of metal dopants were incorporated. Magnetic measurements showed a change in behavior from diamagnetic to paramagnetic with increasing concentration of metal dopants.

The last step in the separation process for a biological product is usually drying, which is the process of thermally removing volatile substances (often water) to yield a solid. In the step preceding drying, the desired product is generally in an aqueous solution and at the desired final level of purity. The most common reason for drying a biological product is that it is susceptible to chemical (e.g., deamidation or oxidation) and/or physical (e.g., aggregation and precipitation) degradation during storage in a liquid formulation. Another common reason for drying is for convenience in the final use of the product. For example, it is often desirable that pharmaceutical drugs be in tablet form. Additionally, drying may be necessary to remove undesirable volatile substances. Also, although many bioproducts are stable when frozen, it is more economical and convenient to store them in dry form rather than frozen. Drying is now an established unit operation in the process industries. However, because most biological products are thermally labile, only those drying processes that minimize or eliminate thermal product degradation are actually used to dry biological products. This chapter focuses on the types of dryer that have generally found the greatest use in the drying of biological products: vacuum-shelf dryers, batch vacuum rotary dryers, freeze dryers, and spray dryers [1]. The principles discussed, however, will apply to other types of dryers as well. We begin with the fundamental principles of drying, followed by a description of the types of dryer most used for biological products. Then we present scale-up and design methods for these dryers. After completing this chapter, the reader should be able to do the following: • Do drying calculations involving relative humidity using the psychrometric moisture chart and the equilibrium moisture curve for the material being dried. • Calculate the relative amounts of bound and unbound water in wet solids before drying. • Model heat transfer in conductive drying and calculate conductive drying times. • Interpret drying rate curves. • Calculate convective drying times of nonporous solids based on mass transfer.

If a product is synthesized intracellularly and not secreted by the producing cell, or if the product is to be extracted from plant, animal, or fungal tissue, it is necessary to remove the product from the cell or tissue by force. The choice of procedure is highly dependent on the nature of the product and the nature of the cell or tissue. It was seen in Chapter 1 that bioproducts represent a wide variety of chemical species. In this chapter, we also see that the sources of bioproducts—cells and tissues—are widely varied. For this reason, there exists a wide variety of methods for breaking, or lysing, cells and tissues, broadly classified as “chemical” and “physical” methods. Once cells have been suspended and/or broken open, the resulting suspension of solids must be separated from the liquid in which it is suspended. This separation process, filtration and/or sedimentation (the subjects of the next two chapters), is enhanced by having larger particles. Larger particles can be achieved by flocculation, a process whereby particles are aggregated into clusters, or flocs. In recent years, it has become desirable to isolate specific cell types from mixtures of suspended cells and to deliver the resulting cell subpopulation(s) to a process for which they, and only they, are required. Most examples come from in vivo sources such as blood and dispersed tissue cells. This aspect of cell processing, namely, cell purification, places special demands on separation processes that are capable of handling particulate matter under conditions that allow cells to remain alive. This chapter presents two major elements of cell processing: the science and engineering of cell rupture by physical and chemical methods and the flocculation of cells and subcellular particles in aqueous suspension. First, however, it is helpful to develop a broad appreciation for the variety and compositions of cells that are likely to be encountered in downstream bioprocessing.

Bioproducts—chemical substances or combinations of chemical substances that are made by living things—range from methanol to whole cells. They are derived by extraction from whole plants and animals or by synthesis in bioreactors containing cells or enzymes. Bioproducts are sold for their chemical activity: methanol for solvent activity, ethanol for its neurological activity or as a fuel, penicillin for its antibacterial activity, taxol for its anticancer activity, streptokinase (an enzyme) for its blood clot dissolving activity, hexose isomerase for its sugar-converting activity, and whole Bacillus thuringiensis cells for their insecticide activity, to name a few very different examples. The wide variety represented by this tiny list makes it clear that bioseparations must encompass a correspondingly wide variety of methods. The choice of separation method depends on the nature of the product, remembering that purity, yield, and activity are the goals, and the most important of these is activity. This first chapter therefore reviews the chemical properties of bioproducts with themes and examples chosen to heighten awareness of those properties that must be recognized in the selection of downstream processes that result in acceptably high final purity while preserving activity. The final part of this chapter is an introduction to the field of bioseparations, which includes a discussion of the stages of downstream processing, the basic principles of engineering analysis as applied to bioseparations, and the various factors involved in developing a bioproduct for the marketplace. The pharmaceutical, agrichemical, and biotechnology bioproduct industries account for many billion dollars in annual sales—neglecting, of course, commodity foods and beverages. By “bioproduct” we mean chemical substances that are produced in or by a biological process, either in vivo or ex vivo (inside or outside a living organism). Figure 1.1 indicates a clear inverse relationship between bioproduct market size and cost. Owing to intense competition, cost, price, and value are very closely related, except in the case of completely new products that are thoroughly protected by patents, difficult to copy, and of added value to the end user.

Filtration is an operation that has found an important place in the processing of biotechnology products. In general, filtration is used to separate particulate or solute components in a fluid suspension or solution according to size by flowing under a pressure differential through a porous medium. There are two broad categories of filtration, which differ according to the direction of the fluid feed in relation to the filter medium. In conventional or dead-end filtration, the fluid flows perpendicular to the medium, which generally results in a cake of solids depositing on the filter medium. In crossflow filtration (which is also called tangential flow filtration), the fluid flows parallel to the medium to minimize buildup of solids on the medium. Conventional and crossflow filtration are illustrated schematically in Figure 4.1. Conventional filtration is typically used when a product has been secreted from cells, and the cells must be removed to obtain the product that is dissolved in the liquid. Antibiotics and steroids are often processed by using conventional filtration to remove the cells. Conventional filtration is also commonly used for sterile filtration in biopharmaceutical production. Crossflow filtration has been used in a wide variety of applications, including the separation of cells from a product that has been secreted, the concentration of cells, the removal of cell debris from cells that have been lysed, the concentration of protein solutions, the exchange or removal of a salt or salts in a protein solution, and the removal of viruses from protein solutions. Filtration often occurs in the early stages of bioproduct purification, in keeping with the process design heuristic “remove the most plentiful impurities first” (see Chapter 12, Bioprocess Design and Economics). At the start of purification, the desired bioproduct is usually present in a large volume of aqueous solution, and it is desirable to reduce the volume as soon as possible to reduce the scale and thus the cost of subsequent processing operations. Filtration, along with sedimentation and extraction (see Chapters 5 and 6), is an effective means of accomplishing volume reduction.

Liquid chromatography and adsorption processes are based on the differential affinity of various soluble molecules for specific types of solids. In these processes, equilibrium is approached between a solid phase, often called the resin, or stationary phase, and the soluble molecules in a liquid phase. The solid phase is “stationary” because it is often packed in a fixed column. Since the liquid phase is often flowing past the solid phase, it is referred to as the mobile phase. Chromatography and adsorption are related unit operations. In chromatography, typically multiple solutes are separated from each other, with the target product solute being one of many that might be recovered at the end of the process step. In adsorption, there are typically only three groups of solutes: those that do not adsorb to the stationary phase (sometimes called “flow through”); secondly, those that adsorb and then are subsequently recovered by an elution step; and thirdly, those solutes that are nearly irreversibly bound and can only be removed from the adsor­bent by regenerating the adsorbent, which usually results in the chemical destruction of these solutes. The word “adsorption” is used both to describe the physical adherence of a solute to a stationary phase, and as the name of the unit operation described above. Adsorption is a subset of the “sorption” phenomena, absorption (transfer of a solute from one phase into another) and ion exchange (exchange of a counter-ion between two opposing co-ions) being the other two sorption phenomena. The unit operations chromatography and adsorption can rely on any of these three sorption processes individually or in combination. In chromatography and adsorption, a mixture of solutes in a feed solution is introduced at the inlet of a column containing the stationary phase and separated into zones of individual solutes over the length of the column. The solutes are carried by the convective action of an elution solvent that is continuously fed to the column after the feed solution has been introduced.

This chapter teaches students and practicing engineers the fundamentals of bioprocess design with an emphasis on bioseparation processes. It combines the information presented in earlier chapters for use in the context of integrated processes. The ultimate objective is to enable the reader to efficiently synthesize and evaluate integrated bioseparation processes. Given a product and a desired annual production rate (process throughput), bioprocess design endeavors to answer the following and other related questions: What are the required amounts of raw materials and utilities needed for a single batch? What is the total amount of resources consumed per year? What is the required size of process equipment and supporting utilities? Can the product be produced in an existing facility or is a new plant required? What is the total capital investment? What is the manufacturing cost? What is the optimum batch size? How long does a single batch take? How much product can be generated per year? Which process steps or resources constitute scheduling and throughput bottlenecks? What changes can increase throughput? What is the environmental impact of the process (i.e., amount and type of waste materials)? Which design is the “best” among several plausible alternatives? After completing this chapter, the reader should be able to do the following: • Initiate a process design and choose the appropriate sequencing of processing steps. • Set up a process flowsheet using the unit procedure concept. • Become familiar with batch process simulators. • Schedule batch processes. • Estimate capital and operating costs. • Perform profitability analysis. • Assess the environmental impact of a process. • Perform process sensitivity analyses. Process design is the conceptual work done prior to building, expanding, or retrofitting a process plant. It consists of two main activities, process synthesis and process analysis. Process synthesis is the selection and arrangement of a set of unit operations (process steps) capable of producing the desired product at an acceptable cost and quality. Process analysis is the evaluation and comparison of different process synthesis solutions.

Evaporation is a process that involves the removal by vaporization of part of the solvent from a solution, with the objective being to concentrate the solution. In the evaporation of solutions containing biological compounds, the volatile solvent can be water or an organic solvent. Organic solvents are frequently used for antibiotics, steroids, and peptides. Often the solution is under a moderate vacuum, at pressures down to about 0.05 atm absolute [1], which is especially important for heat-sensitive biologicals where the temperature should be as low as possible to minimize degradation. The energy source for evaporation is usually steam at a low pressure, below 3 atm absolute [1]. Evaporation processes typically occur after the processes used for the removal of insolubles. They are often used to concentrate a solution just prior to the bioproduct being crystallized or precipitated. Evaporation can often be coupled with extraction: for example, a bioproduct is extracted from an aqueous stream with an organic solvent, and the extract is sent to an evaporator for concentration. In this chapter, the basic principles of evaporation are discussed, followed by a description of the most common types of evaporators for heat sensitive biological products and a discussion of scale-up and design methods. After completing this chapter, the reader should be able to do the following: • Explain the different types of resistances to heat transfer in an evaporator. • Take into account the boiling point elevation in heat transfer calculations for evaporators. • Calculate the heat transfer resistances and residence time for the concentration of a heat-sensitive bioproduct in a falling film evaporator. • Estimate the fouling factor in an evaporator. • Calculate the maximum allowable vapor velocity from an evaporator. • Select an appropriate type of evaporator to use based on the specific operational and product characteristics. • Size evaporators based on specific operating conditions and the expected overall heat transfer coefficient. The main principles to consider for evaporators are heat transfer and vapor-liquid separation. The theoretical basis of these principles will be discussed.

Designed for undergraduates, graduate students, and industry practitioners, Bioseparations Science and Engineering fills a critical need in the field of bioseparations. Current, comprehensive, and concise, it covers bioseparations unit operations in unprecedented depth. In each of the chapters, the authors use a consistent method of explaining unit operations, starting with a qualitative description noting the significance and general application of the unit operation. They then illustrate the scientific application of the operation, develop the required mathematical theory, and finally, describe the applications of the theory in engineering practice, with an emphasis on design and scaleup. Unique to this text is a chapter dedicated to bioseparations process design and economics, in which a process simular, SuperPro Designer® is used to analyze and evaluate the production of three important biological products. New to this second edition are updated discussions of moment analysis, computer simulation, membrane chromatography, and evaporation, among others, as well as revised problem sets. Unique features include basic information about bioproducts and engineering analysis and a chapter with bioseparations laboratory exercises. Bioseparations Science and Engineering is ideal for students and professionals working in or studying bioseparations, and is the premier text in the field.