Bolton-Hunter Reagent Research Articles

In Vitro Cytotoxicity of Trastuzumab (Tz) and Se-Trastuzumab (Se-Tz) against the Her/2 Breast Cancer Cell Lines JIMT-1 and BT-474.

Her/2+ breast cancer accounts for ~25% mortality in women and overexpression of Her/2 leads to cell growth and tumor progression. Trastuzumab (Tz) with Taxane is the preferred treatment for Her/2+ patients. However, Tz responsive patients often develop resistance to Tz treatment. Herein, redox selenides (RSe-) were covalently linked to Tz using a selenium (Se)-modified Bolton–Hunter Reagent forming Seleno-Trastuzumab (Se-Tz; ~25 µgSe/mg). Se-Tz was compared to Tz and sodium selenite to assess the viability of JIMT-1 and BT-474 cells. Comparative cell viability was examined by microscopy and assessed by fluorometric/enzymatic assays. Se-Tz and selenite redox cycle producing superoxide (O2•−) are more cytotoxic to Tz resistant JIMT-1 and Tz sensitive BT-474 cells than Tz. The results of conjugating redox selenides to Tz suggest a wider application of this technology to other antibodies and targeting molecules.

In vitro Cytotoxic Effects of Control and Selenium Conjugated Transferrins against Leukemia Cell Lines K562 and THP-1

In vitro Cytotoxic Effects of Control and Selenium Conjugated Transferrins against LeHuman leukemia is a malignancy of hematopoietic stem cells with impaired differentiation and uncontrolled proliferation. Many leukemia cell lines over-express the trans-membrane transferrin receptor (TfR) which endocytoses iron (Fe) bound Holo-transferrin (Holo-Tf). Over-expression of the TfR has permitted transferrin-mediated drug delivery targeting leukemia cells. Selenides (RSe-), can generate free radicals, i.e.; superoxide (O2.-) and other Reactive Oxygen Species (ROS) by redox cycling; oxidizing thiols (RSH) within cells following endocytosis. Superoxide generation by selenium (Se) is reported to cause cell apoptosis/necrosis due to increased endogenous oxidative stress. In the present study, Se as ROCH2CH2SeCN was covalently attached to Lysine residues of Apo-, Holo-Tf, and Human Serum Albumin (HuSA) using a Se-modified Bolton-Hunter reagent. The Se conjugated proteins were used to target the TfR and experimentally treat K562 and THP-1 leukemia cell lines. Cells were also treated with sodium selenite (10 μgSe/well) as a positive control known to cause cell death.ukemia Cell Lines K562 and THP-1.

Two-step enzymatic strategy for the synthesis of a smart phenolic polymer and further immobilization of a β-galactosidase able to catalyze transglycosydation reaction

A rapid and efficient enzymatic procedure for the preparation of an immobilized β-galactosidase has been described. In a first step, soybean peroxidase was used to catalyze the polymerization of a strategically activated phenol (N–Succinimidyl 3–(4–hydroxyphenyl)propionate, known as Bolton-Hunter reagent). The phenolic support was directly employed for immobilizing β–galactosidase from Bacillus circulans (ATCC 31382, β–Gal–3), giving rise to a new biocatalyst subsequently applied in the synthesis of a β–galatodisaccharide (Gal–β(1–3)–GlcNAc and Gal–β(1–3)–GalNAc). The reaction proceeded with high conversion rates and total regioselectivity. Reusability assays were performed with the same reaction conditions finding that the immobilized enzyme retains about 55% of its activity after eight batches. Finally and based on our results, the two-step enzymatic procedure presented here is a good and green alternative to the preparation of carbohydrates with biological activities.

Radiolabeling of melatonin using different oxidizing agents for immunoassay purpose

An efficient method was developed for radioiodination of melatonin with 125I. Chloramine-T and iodogen were used as oxidizing agents. The radioiodination was carried out via electrophilic substitution of the H atom with the I+ cation. Indirect iodination using Bolton–Hunter reagent was also performed. The influence exerted on the radiochemical yield by the melatonin concentration, pH of the reaction mixture, type of oxidizing agent, and reaction time was studied. The shelf life and specific activity of the product were determined. The radiochemical yield and product purity were determined by HPLC and paper electrophoresis. The immunoreactivity of the tracer was checked using its specific antibody. Specific and sensitive polyclonal antimelatonin was prepared using two New Zealand rabbits, which were injected with 5-methoxy-N-acetyltryptamine–bovine serum albumin conjugate as immunogen. The tracer prepared by the indirect method with iodogen had high specific activity, high yield, and long shelf life; it was also more selective in the melatonin assessment using radioimmunoassay technique.

Brain and Organ Uptake in the Rhesus Monkey in Vivo of Recombinant Iduronidase Compared to an Insulin Receptor Antibody-Iduronidase Fusion Protein.

Mucopolysaccharidosis type I (MPSI) is caused by mutations in the gene encoding the lysosomal enzyme, α-l-iduronidase (IDUA), and patients with MPSI are currently treated with IDUA enzyme replacement therapy (ERT). However, the majority of MPSI patients have severe CNS involvement, and conventional ERT does not treat the brain. The failure of ERT to treat the brain is believed to be due to the lack of IDUA transport through the blood-brain barrier (BBB). However, BBB transport of IDUA has not been directly measured, to date. BBB transport of IDUA may be enhanced by fusion of the enzyme to a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb binds the insulin receptor on the BBB to trigger transport into the brain and acts as a molecular Trojan horse to deliver IDUA to brain cells. Therefore, the purpose of the present investigation was to compare, side-by-side, the BBB transport of IDUA alone and the HIRMAb-IDUA fusion protein in the Rhesus monkey in vivo. Each protein was radio-iodinated by conjugation with the [125I]-Bolton-Hunter reagent and injected intravenously (IV) in the primate. The uptake by brain, and peripheral organs, was measured by whole body autoradiography. The results show there is no transport of IDUA alone into the brain, but that the brain uptake of the HIRMAb-IDUA fusion protein is high, 1.2% injected dose/brain. There is comparable uptake of the IDUA and the HIRMAb-IDUA fusion protein by peripheral organs, where uptake is primarily controlled by the mannose 6-phosphate receptor. The work suggests that treatment of MPSI with the HIRMAb-IDUA fusion protein will be as effective as IDUA in peripheral organs, but offer the benefit of treatment of the central nervous system in MPSI.

Insulin Receptor Antibody-α-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts.

Mucopolysaccharidosis Type IIIB (MPSIIIB) is caused by mutations in the gene encoding the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU). MPSIIIB presents with severe disease of the central nervous system, but intravenous NAGLU enzyme replacement therapy has not been developed because the NAGLU enzyme does not cross the blood-brain barrier (BBB). A BBB-penetrating form of the enzyme was produced by re-engineering NAGLU as an IgG-enzyme fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb traverses the BBB via transport on the endogenous insulin receptor and acts as a molecular Trojan horse to ferry the fused NAGLU across the BBB from blood. The NAGLU was fused to the carboxyl terminus of each heavy chain of the HIRMAb via an extended 31-amino acid linker, and the fusion protein is designated HIRMAb-LL-NAGLU. The fusion protein retains high affinity binding to the HIR, and on a molar basis has an enzyme activity equal to that of recombinant human NAGLU. Treatment of MPSIIIB fibroblasts with the fusion protein normalizes intracellular NAGLU enzyme activity and reduces sulfate incorporation into intracellular glycosoaminoglycan. The fusion protein is targeted to the lysosomal compartment of the cells as shown by confocal microscopy. The fusion protein was radiolabeled with the [(125)I]-Bolton-Hunter reagent and injected intravenously in the adult Rhesus monkey. The fusion protein was rapidly cleared from plasma by all major peripheral organs. The high brain uptake of the fusion protein, 1% injected dose/brain, enables normalization of brain NAGLU enzyme activity with a therapeutic dose of 1 mg/kg. The HIRMAb-LL-NAGLU fusion protein is a new treatment of the brain in MPSIIIB, which can be administered by noninvasive intravenous infusion.

Insulin receptor antibody-sulfamidase fusion protein penetrates the primate blood-brain barrier and reduces glycosoaminoglycans in Sanfilippo type A cells.

Mutations in the lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (SGSH), also called sulfamidase, cause accumulation of lysosomal inclusion bodies in the brain of children born with mucopolysaccharidosis type IIIA, also called Sanfilippo type A syndrome. Enzyme replacement therapy with recombinant SGSH does not treat the brain because the enzyme is a large molecule drug that does not cross the blood–brain barrier (BBB). A BBB-penetrating form of SGSH was produced by re-engineering the enzyme as an IgG fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb domain of the HIRMAb–SGSH fusion protein acts as a molecular Trojan horse to ferry the fused enzyme across the BBB. The HIRMAb–SGSH was produced in stably transfected host cells and purified to homogeneity by protein A chromatography. The fusion protein reacted with antibodies against either human IgG or SGSH on Western blotting. High affinity binding to the HIR was retained following SGSH fusion to the HIRMAb, with an EC50 of 0.33 ± 0.05 nM in an HIR binding ELISA. The SGSH enzyme activity of the HIRMAb–SGSH fusion protein was 4712 ± 388 units/mg protein based on a two-step fluorometric enzyme assay. The HIRMAb–SGSH was taken up by lysosomes in MPSIIIA fibroblasts, and treatment of these cells with the fusion protein caused an 83% reduction in sulfate incorporation into lysosomal glycosoaminoglycans. The HIRMAb–SGSH fusion protein was radiolabeled with the [125I]-Bolton–Hunter reagent and injected intravenously in the Rhesus monkey. The brain uptake of the fusion protein was high, ∼1% injected dose/brain. Calculations, based on this level of brain uptake, suggest normalization of brain SGSH enzyme activity is possible following administration of therapeutic doses of the fusion protein. These studies describe a novel IgG–SGSH fusion protein that is a new noninvasive treatment of the brain in MPS type IIIA.

Blood-Brain Barrier Molecular Trojan Horse Enables Imaging of Brain Uptake of Radioiodinated Recombinant Protein in the Rhesus Monkey

Recombinant proteins are large molecule drugs that do not cross the blood-brain barrier (BBB). However, BBB-penetration of protein therapeutics is enabled by re-engineering the recombinant protein as IgG fusion proteins. The IgG domain is a monoclonal antibody (mAb) against an endogenous BBB receptor-mediated transport system, such as the human insulin receptor (HIR), and acts as a molecular Trojan horse to ferry the fused protein across the BBB. In the present study, a recombinant lysosomal enzyme, iduronate 2-sulfatase (IDS), is fused to the HIRMAb, and BBB penetration of the IDS alone vs the HIRMAb-IDS fusion protein is compared in the Rhesus monkey. Recombinant IDS and the HIRMAb-IDS fusion protein were radiolabeled with indirect iodination with the [(125)I]-Bolton-Hunter reagent and with direct iodination with Iodogen/[(125)I]-idodine. IDS and the HIRMAb-IDS fusion protein have comparable plasma pharmacokinetics and uptake by peripheral organs. IDS does not cross the BBB. The HIRMAb-IDS fusion protein crosses the BBB and the brain uptake is 1% of injected dose/brain. Brain imaging shows HIRMAb-IDS penetration to all parts of brain, and immunoprecipitation of brain radioactivity shows intact fusion protein in brain. The use of BBB molecular Trojan horses enables brain imaging of recombinant proteins that are re-engineered for BBB transport.

Pharmacokinetics and brain uptake in the rhesus monkey of a fusion protein of arylsulfatase a and a monoclonal antibody against the human insulin receptor

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder of the brain caused by mutations in the gene encoding the lysosomal sulfatase, arylsulfatase A (ASA). It is not possible to treat the brain in MLD with recombinant ASA, because the enzyme does not cross the blood-brain barrier (BBB). In the present investigation, a BBB-penetrating IgG-ASA fusion protein is engineered and expressed, where the ASA monomer is fused to the carboxyl terminus of each heavy chain of an engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor-mediated transport on the endogenous BBB insulin receptor, and acts as a molecular Trojan horse to ferry the ASA into brain from blood. The HIRMAb-ASA is expressed in stably transfected Chinese hamster ovary cells grown in serum free medium, and purified by protein A affinity chromatography. The fusion protein retains high affinity binding to the HIR, EC50 = 0.34 ± 0.11 nM, and retains high ASA enzyme activity, 20 ± 1 units/mg. The HIRMAb-ASA fusion protein is endocytosed and triaged to the lysosomal compartment in MLD fibroblasts. The fusion protein was radio-labeled with the Bolton-Hunter reagent, and the [(125) I]-HIRMAb-ASA rapidly penetrates the brain in the Rhesus monkey following intravenous administration. Film and emulsion autoradiography of primate brain shows global distribution of the fusion protein throughout the monkey brain. These studies describe a new biological entity that is designed to treat the brain of humans with MLD following non-invasive, intravenous infusion of an IgG-ASA fusion protein.

A Facile Way to Tune Mechanical Properties of Artificial Elastomeric Proteins-Based Hydrogels

Protein-based hydrogels have attracted considerable interests due to their potential applications in biomedical engineering and material sciences. Using a tandem modular protein (GB1)(8) as building blocks, we have engineered chemically cross-linked hydrogels via a photochemical cross-linking strategy, which is based on the cross-linking of two adjacent tyrosine residues into dityrosine adducts. However, because of the relatively low reactivity of tyrosine residues in GB1, (GB1)(8)-based hydrogels exhibit poor mechanical properties. Here, we report a Bolton-Hunter reagent-based, facile method to improve and tune the mechanical properties of such protein-based hydrogels. Using Bolton-Hunter reagent, we can derivatize lysine residues with phenolic functional groups to modulate the phenolic (tyrosine-like) content of (GB1)(8). We show that hydrogels made from derivatized (GB1)(8) with increased phenolic content show significantly improved mechanical properties, including improved Young's modulus, breaking modulus as well as reduced swelling. These results demonstrate the great potential of this derivatization method in constructing protein-based biomaterials with desired macroscopic mechanical properties.

Microwave-assisted One-pot Synthesis of <em>N</em>-succinimidyl-4-[<sup>18</sup>F]fluorobenzoate ([<sup>18</sup>F]SFB)

Biomolecules, including peptides¹⁻⁹, proteins¹⁰⁻¹¹, and antibodies and their engineered fragments¹²⁻¹⁴, are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.¹⁵⁻¹⁸ Therefore, significant effort has devoted to the synthesis and exploration of ¹⁸F-labeled biomolecules. Although there are elegant examples of the direct ¹⁸F-labeling of peptides,¹⁹⁻²² the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.²³(,)²⁴ N-Succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB),²⁵⁻³⁷ a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the ¹⁸F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [¹⁸F]SFB, the resulting [F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.¹ Most [¹⁸F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [¹⁸F]SFB syntheses have been reported²⁹⁻³²,⁴¹⁻⁴² they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [¹⁸F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process. Herein, we report a concise preparation of [¹⁸F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,³⁸⁻⁴¹ can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.³⁸Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step. ²⁸(,)⁴³The novelty of our improved [¹⁸F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [¹⁸F]SFB.

Abstract SY20-03: Micelle-based targeting radio-nanotheranostic agents against cancer

Abstract We have developed a novel amphiphilic polymer (telodendrimer), comprised of linear polyethylene glycol and cholic acid cluster, that can self-assemble under aqueous condition to form stable micelles. We have succeeded in loading the nanoparticles with several different hydrophobic drugs. These include paclitaxel, etoposide, vincristine, doxorubicin, bortezomib, sorafenib, lapatinib, temsirolimus, and dexamethasone. This nanoplatform is highly versatile. Particle size can be readily tuned by varying the configuration and composition of the telodendrimer. Nanoparticles of various sizes loaded with DiD, a near infrared hydrophobic dye, were prepared and their biodistribution evaluated in nude mice bearing ovarian cancer xenografts. Particles smaller than 60 nm preferentially targeted the tumors whereas particles at 150 nm were found to concentrate at the liver and lung but with low tumor uptake. The surface chemistry can be easily modified by tethering various acidic or basic peptides to the distal polyethylene glycol tail of the telodendrimers. Undesirable liver uptake was very high for those nanoparticles with high surface charges, either positive or negative. In contrast, liver uptake was very low but tumor uptake was very high when the surface charge was slightly negative. Ligation of tumor targeting ligands to the particle surface further improved tumor targeting and intracellular uptake at the tumor site, resulting in better therapeutic efficacy in xenograft model. In addition to xenograft models, we have also demonstrated that the nanoparticles could preferentially target early spontaneous mammary tumors of PyV-MT transgenic mice. We have just completed a phase I study of placiltaxel-loaded nanoparticles in companion dogs with lymphoma and showed that the drug is safe. Maximum tolerated dose (MTD) was determined to be 97mg/meter square. A phase II study of the same nanoformulation in companion dogs with solid tumor is ongoing. The nanoparticles are stable both in vitro and in vivo. Additional stability of the nanoparticles can be achieved by chemical stitching of adjacent telodendrimers with disulfide bonds after drug loading. Such crosslinked nanoparticles are extremely stable, even in the presence of sodium dodecyl sulfate, a strong ionic detergent. Drug release from crosslinked nanoparticles is much slower than that from non-crosslinked nanoparticles. However, upon addition of 10 mM of glutathione, drug release rate from the crosslinked nanoparticles paralleled that of the non-crosslinked nanoparticles. Since glutathione level is high at the tumor site and inside the tumor cells, it is expected that the disulfide bonds of such crosslinked nanoparticles will be reduced after taken up by tumor cells. Therapeutic studies in xenograft model indeed confirmed that crosslinked paclitaxel-loaded nanoparticle was more efficacious than non-crosslinked nanoparticles. For radioimaging studies, the lysine side chain of telodendrimers was radioiodinated with [I-125]-labeled Bolton-Hunter reagent and then loaded with paclitaxel to form stable [I-125]-labeled nanoparticles. MicroCT/SPECT imaging study with this novel radioprobe in nude mice bearing ovarian cancer xenograft revealed gradual uptake of the radioprobe by the tumor within the first 24 hours. At the 24-hour timepoint, significant amount of radioprobe remained in the circulation. However, by 48 hours, most of the circulating radioprobe disappeared while tumor uptake remained high as late as 94 hours. Liver, spleen, lung and renal uptake were minimal. Together, these studies suggest that an optimal therapeutic nanoparticle should be less than 60 nm in diameter, have slightly negative surface charge, display cancer targeting and pro-endocytotic ligand(s), and be cross-linked by disulfide bonds after drug loading. Such nanoparticle drug is expected to have minimal premature drug release, be preferentially taken up by solid tumors via the enhanced permeability (EPR) effect, enter the tumor cells via the tumor cell targeting ligands, and drug released inside the tumor cells where the glutathione level is high. The addition of an imaging radionuclide to such nanoparticle will make it a highly effective radio-nanotheranostic agent against cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY20-03. doi:10.1158/1538-7445.AM2011-SY20-03

Radioiodination and biodistribution of quantum dots using Bolton–Hunter reagent

In this study, the radioiodination and biodistribution of quantum dots (QDs) using Bolton-Hunter reagent were investigated. Radioiodination yield was 33.4 ± 2.0%. Fluorescent intensity of radioiodinated QDs decreased to 75.4% of the maximum prior to radioiodination. In biodistribution and ex vivo fluorescence imaging, radioiodinated QDs were highly accumulated in reticuloendothelial system (liver and spleen) and had low level bone uptakes and slow clearance from body. These results suggest that the radioiodination method of nanoparticles using Bolton-Hunter reagent could be easily used in the biodistribution and quantification of nanoparticles in vivo.

γ-Glutamyl PAMAM Dendrimer as Versatile Precursor for Dendrimer-Based Targeting Devices

Poly(amidoamine) (PAMAM) dendrimers are highly branched spherical polymers that have a unique surface of primary amine groups and provide a versatile design for targeted delivery of pharmaceuticals and imaging agents. Acetylation or succinylation of surface amine groups of PAMAM dendrimer derivatives is frequently performed to reduce nonspecific uptake. However, since targeting molecules, drugs/imaging agents, and acylating reagents react with the amine groups on dendrimer, such modification may limit the number of targeting molecules and/or drugs or may result in insufficient charge reduction. In this study, a gamma-glutamyl PAMAM dendrimer was designed and synthesized as a new precursor for targeting device. The relationship between surface electrical properties of the PAMAM dendrimer derivatives and pharmacokinetics was also determined. A PAMAM dendrimer (generation 4.0) was modified with a small number of Bolton-Hunter reagent to prepare Phe-P (pI 9.2). The amine residues of Phe-P were gamma-glutamylated to prepare Glu-P (pI 7.1). The alpha-amine residues of Glu-P were then acetylated or succinylated to prepare Ac-Glu-P (pI 5.3) or Suc-Glu-P (pI 3.6). For comparison, Phe-P was acetylated or succinylated to prepare Ac-P (pI 6.0) or Suc-P (pI 5.1). All the PAMAM dendrimer derivatives exhibited similar molecular size (7.2 to 7.8 nm) except for Ac-P (5.1 nm). The biodistribution studies were performed after radioiodination of each PAMAM dendrimer derivative with Na[(125)I]I. When injected intravenously to mice, both [(125)I]Ac-P and [(125)I]Suc-P exhibited prolonged radioactivity levels in the blood and significantly lower hepatic and renal radioactivity levels than those of [(125)I]Phe-P. Both [(125)I]Glu-P and [(125)I]Ac-Glu-P showed residence times in the blood similar to those of [(125)I]Ac-P and [(125)I]Suc-P. However, [(125)I]Glu-P also registered higher radioactivity levels in the kidney. High hepatic and renal radioactivity levels were observed with highly anionic [(125)I]Suc-Glu-P. These results indicate that, while the manipulation of pI between 5 to 6 would be appropriate to enhance blood retention and reduce renal and hepatic uptake, the amount of primary amine residues on dendrimer surface may also play a crucial role in their renal uptake. The findings in this study show that gamma-glutamyl PAMAM dendrimers would constitute versatile precursors to prepare PAMAM dendrimer-based targeting devices due to their neutral molecular charge (pI 7.1) and the presence of a large number of alpha-amine residues available for conjugation of targeting molecules and drugs/imaging agents.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens α-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.

Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein-2

This work evaluated gelatin microparticles and biodegradable composite scaffolds for the controlled release of bone morphogenetic protein-2 (BMP-2) in vitro and in vivo. Gelatin crosslinking (10 and 40 mM glutaraldehyde), BMP-2 dose (6 and 60 ng BMP-2 per mg dry microparticles), buffer type (phosphate buffered saline (PBS) and collagenase-containing PBS), and gelatin type (acidic and basic) were investigated for their effects on BMP-2 release. Release profiles were also observed using poly(lactic- co-glycolic acid) (PLGA) microparticles with varying molecular weights (8300 and 57,500). In vitro and in vivo studies were conducted using radiolabeled BMP-2; the chloramine-T method was preferred over Bolton–Hunter reagent for radioiodination with this system. BMP-2 release from PLGA microparticles resulted in a moderate burst release followed by minimal cumulative release, while BMP-2 release from gelatin microparticles exhibited minimal burst release followed by linear release kinetics in vitro. Growth factor dose had a small effect on its normalized release kinetics probably because of an equilibrium between gelatin-bound and unbound BMP-2. Differences in release from acidic and basic gelatin microparticles may result from the different pretreatment conditions used for gelatin synthesis. The in vitro release kinetics for both gelatin microparticles alone and within composite scaffolds were dependent largely on the extent of gelatin crosslinking; varying buffer type served to confirm that controlled release relies on enzymatic degradation of the gelatin for controlled release. Finally, in vivo studies with composite scaffolds exhibited minimal burst and linear release up to 28 days. In summary, dose effects on BMP-2 release were found to be minimal while varying gelatin type and release medium can alter release kinetics. These results demonstrate that a systematic control of BMP-2 delivery from gelatin microparticles can be achieved by altering the extent of basic gelatin crosslinking.

Doubly radiolabeled liposomes for pretargeted radioimmunotherapy

The aim of this study was to design liposomes as radioactivity carriers for pretargeted radioimmunotherapy with favorable pharmacokinetic parameters. To monitor the liposomes integrity in vivo, their surface was radiolabeled with indium-111 bound to DTPA-derivatized phosphatidylethanolamine (DSPE-DTPA) and the aqueous phase was labeled by using an original active loading technique of radioiodinated Bolton–Hunter reagent (BH) that reacts with pre-encapsulated arginine to form a positively charged conjugate ( 125I-BH-arginine). Different formulations of doubly radiolabeled liposomes were tested in vitro and in vivo to evaluate radiolabeling stability, integrity of the vesicles and their pharmacokinetics. Radiolabeling yields were high (surface >75%, encapsulation >60%) and stable (>85% after 24 h in serum 37 °C). In vivo, the pharmacokinetic behavior of doubly radiolabeled liposomes was strongly dependant on the formulation. Blood clearance of PEGylated liposomes (DSPC/Chol/DSPE-DTPA/DSPE-PEG5%) was 0.15 mL/h compared to a conventional formulation (DSPC/Chol/DSPE-DTPA: clearance 1.44 mL/h). Non-encapsulated BH-arginine conjugate was quickly eliminated in urine (clearance 6.04 mL/h). Blood kinetics of the two radionuclides were similar and radiochromatographic profiles of mice serum confirmed the integrity of circulating liposomes. The significant reduction of activity uptake in organs after liposome catabolism (liver and spleen), achieved by the rapid renal elimination of 125I-BH-arginine, should bring significant improvements for targeted radionuclide therapy with sterically-stabilized liposomes.

P2X5 Subunit Assembly Requires Scaffolding by the Second Transmembrane Domain and a Conserved Aspartate

Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.

Solution Radioimmunoassay of Proteins and Peptides

This unit describes a solution-phase radioimmunoassay (RIA) using the double antibody/PEG method. A solution RIA determines the amount of a specific protein (or peptide) in a sample by comparison to a standard curve of the same protein (or peptide). The amount of protein (or peptide) in the sample is determined based on its ability to compete with a radiolabeled tracer for binding to a specific antibody. A secondary antibody and PEG are used to precipitate the primary antibody-protein complexes. The amount of radioactivity in the complex is inversely proportional to the amount of protein in the sample. Under ideal conditions, an RIA is capable of measuring picograms of a protein (or peptide), but only if the tracer is labeled at a high specific activity. To this end, this unit also presents protocols for radioiodination of proteins and peptides using lactoperoxidase or the Bolton-Hunter reagent.