Length Of Spacer Arm Research Articles

The spacer arm length in cell-penetrating peptides influences chitosan/siRNA nanoparticle delivery for pulmonary inflammation treatment.

Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of R9Gn-chitosan/siRNA nanoparticles were investigated in vitro. Increasing the spacing arm length did not significantly affect the complex formation between R9Gn-chitosan and siRNA. However, R9G10-chitosan was much more effective in delivering genes both in vitro and in vivo compared with non-modified chitosan (without the peptide) and R9-chitosan (without the spacer arm). Chitosan derivatives modified with oligoarginine containing a spacer arm can be considered as potential delivery vehicles for various genes.

In Silico Study of Spacer Arm Length Influence on Drug Vectorization by Fullerene C60

This work studies theoretically the effect of spacer arm lengths on the characteristics of a fullerene C60‐based nanovector. The spacer arm is constituted of a carbon chain including a variable number of methylene groups (n = 2–11). To improve the ability of the fullerene carriage, two arms are presented simultaneously through a malonyl bridge. Then the evolution of selected physicochemical parameters is monitored as a function of the spacer arm length and the angle between the two arms. We show here that while the studied characteristics are almost independent of the spacer arm length or vary monotonically with it, the dipole moment and its orientation vary periodically with the parity of the number of carbon atoms. This periodicity is related to both modules and orientations of dipole moments of the spacer arms. In the field of chemical synthesis, these results highlight the importance of theoretical calculations for the optimization of operating conditions. In the field of drug discovery, they show that theoretical calculations of the chemical properties of a drug candidate can help predict its in vivo behaviour, notably its bioavailability and biodistribution, which are known to be tightly dependent of its polarity.

On the efficiency of NHS ester cross-linkers for stabilizing integral membrane protein complexes.

We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC(2)D(2). We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.

Recovery of small dye molecules from aqueous solutions using charged ultrafiltration membranes

Recovery of reactive dyes from effluent streams is a growing environmental challenge. In this study, various charged regenerated cellulose (RC) ultrafiltration (UF) membranes were prepared and tested for removal of three model reactive dyes (reactive red ED-2B, reactive brilliant yellow K-6G, and reactive brilliant blue KN-R). Data were obtained with charged UF membranes having different spacer arm lengths between the base cellulose and the charge functionality. The effects of charge density of the dye molecules, ionic strength of the feed solution, spacer arm length of charged membranes and filtrate flux were studied. Results indicated that dye retention was greatest with the most negatively charged dye molecule. Higher rejection was also observed in low ionic strength solutions. Results were consistent with model calculations based on the partitioning of a charged sphere into a charged cylindrical pore. The membranes with longer spacer arm length had higher rejection coefficients, consistent with the greater negative charge on these membranes. This study confirms that charged UF membranes can effectively recover small reactive dye molecules at low pressures (below 100kPa) under appropriate solution conditions due to the strong electrostatic repulsion from the membrane pores.

Effect of aliphatic diamine length functionalized silica coated iron magnetic nanoparticles on metal capacity and speed of Cu(II) uptake from aqueous media.

Three solid phase extractors based on silica coated magnetic nanoparticles (Fe3O4-SiO2) functionalized aliphatic diamines were synthesized. The magnetic core of Fe3O4 was synthesized first by the sol-gel method, then protected by a layer of silica using tetraethylorthosilicate (TEOS) followed by functionalization using three aliphatic diamines: 1,2-ethanediamine (1,2-EDA), 1,5-pentanediamine (1,5-PDA) and 1,8-octanediamine (1,8-ODA). These steps for producing the targeted three adsorbents Fe3O4-SiO2-1,2-EDA, Fe3O4-SiO2-1,5-PDA and Fe3O4-SiO2-1,8-ODA with different amines spacer arm were followed up using Fourier Transform Infrared (FTIR) spectra and scanning electron microscope (SEM). The effect of the spacer arm length of the diamines on selective binding and metal capacity values of Cu(II) ions was studied as a function of the time needed to attain equilibrium under optimized conditions of pH, mass of adsorbent and initial concentration of Cu(II) ions. The order of increasing metal capacity for copper ions using magnetic nano-adsorbents was Fe3O4-SiO2-1,2-EDA < Fe3O4-SiO2-1,5-PDA < Fe3O4-SiO2-1,8-ODA at 5 s equilibrium time.

Conserved Walker A Cysteines 431 and 1074 in Human P-Glycoprotein Are Accessible to Thiol-Specific Agents in the Apo and ADP-Vanadate Trapped Conformations

P-Glycoprotein (P-gp) is an ATP-binding cassette efflux transporter involved in the development of multidrug resistance in cancer cells. Although the mechanism of P-gp efflux has been extensively studied, aspects of its catalytic and transport cycle are still unclear. In this study, we used conserved C431 and C1074 in the Walker A motif of nucleotide-binding domains (NBDs) as reporter sites to interrogate the interaction between the two NBDs during the catalytic cycle. Disulfide cross-linking of the C431 and C1074 residues in a Cys-less background can be observed in the presence of M14M and M17M cross-linkers, which have spacer arm lengths of 20 and 25 Å, respectively. However, cross-linking with both cross-linkers was prevented in the ADP-vanadate trapped (closed) conformation. Both C431 and C1074 alone or together (double mutant) in the apo and closed conformations were found to be accessible to fluorescein 5-maleimide (FM) and methanethiosulfonate derivatives of rhodamine and verapamil. In addition, C1074 showed 1.4- and 2-fold higher degrees of FM labeling than C431 in the apo and closed conformations, respectively, demonstrating that C1074 is more accessible than C431 in both conformations. In the presence of P-gp substrates, cross-linking with M17M is still observed, suggesting that binding of substrate in the transmembrane domains does not change the accessibility of the cysteines in the NBDs. In summary, the cysteines in the Walker A motifs of NBDs of human P-gp are differentially accessible to thiol-specific agents in the apo and closed conformations.

Preparation and characterization of fluorophenylboronic acid-functionalized monolithic columns for high affinity capture of cis-diol containing compounds

Boronic acids are important ligands for the selective recognition and capture of cis-diol containing compounds, such as nucleosides and glycoproteins. In a recent study, it was found that 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) exhibited an ultrahigh boronate affinity for binding with monosaccharides. Herein three DFFPBA-functionalized monolithic columns with varying spacer arms were synthesized and characterized. Different cis-diol containing compounds were used for the evaluation of the boronate affinity of the DFFPBA-functionalized monoliths. The DFFPBA-functionalized monoliths exhibited advantageous characteristics. These monoliths exhibited an ultrahigh boronate affinity toward cis-diol containing compounds. Moreover, the monolith with appropriate spacer arm exhibited a low binding pH (6.0) for cis-diols of small molecular weight. These advantages made DFFPBA-functionalized monoliths suitable for the enrichment of trace cis-diol containing compounds in neutral and weak acidic real samples. In addition, it was interesting that the length of spacer arms strongly influenced the boronate affinity: increasing the spacer arm resulted in apparently reduced boronate affinity, and inappropriate spacer arm length even eliminated the boronate affinity toward glycoprotein. To explain such a phenomenon, a possible mechanism was proposed. Finally, the potential of DFFPBA-functionalized monoliths for real applications was demonstrated with the selective enrichment of modified nucleosides from human urine.

Surface‐Imprinted Nanofilaments for Europium‐Amplified Luminescent Detection of Fluoroquinolone Antibiotics

The development and characterization of novel, molecularly imprinted polymer nanofilament-based optical sensors for the analysis of enrofloxacin, an antibiotic widely used for human and veterinary applications, is reported. The polymers were prepared by nanomolding in porous alumina by using enrofloxacin as the template. The antibiotic was covalently immobilized on to the pore walls of the alumina by using different spacers, and the prepolymerization mixture was cast in the pores and the polymer synthesized anchored onto a glass support through UV polymerization. Various parameters affecting polymer selectivity were evaluated to achieve optimal recognition, namely, the spacer arm length and the binding solvent. The results of morphological characterization, binding kinetics, and selectivity of the optimized polymer material for ENR and its derivatives are reported. For sensing purposes, the nanofilaments were incubated in solutions of the target molecule in acetonitrile/HEPES buffer (100 mM, pH 7.5, 50:50, v/v) for 20 min followed by incubation in a 10 mM solution of europium(III) ions to generate a europium(III)-enrofloxacin complex on the polymer surface. The detection event was based on the luminescence of the rare-earth ion (λexc=340 nm; λem=612 nm) that results from energy transfer from the antibiotic excited state to the metal-ion emitting excited state. The limit of detection of the enrofloxacin antibiotic was found to be 0.58 μM.

Understanding chemical reactivity for homo‐ and heterobifunctional protein cross‐linking agents

Chemical cross-linking, combined with mass spectrometry, has been applied to map three-dimensional protein structures and protein-protein interactions. Proper choice of the cross-linking agent, including its reactive groups and spacer arm length, is of great importance. However, studies to understand the details of reactivity of the chemical cross-linkers with proteins are quite sparse. In this study, we investigated chemical cross-linking from the aspects of the protein structures and the cross-linking reagents involved, by using two structurally well-known proteins, glyceraldehyde 3-phosohate dehydrogenase and ribonuclease S. Chemical cross-linking reactivity was compared using a series of homo- and hetero-bifunctional cross-linkers, including bis(sulfosuccinimidyl) suberate, dissuccinimidyl suberate, bis(succinimidyl) penta (ethylene glycol), bis(succinimidyl) nona (ethylene glycol), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, 2-pyridyldithiol-tetraoxaoctatriacontane-N-hydrosuccinimide and succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol]ester. The protein structure itself, especially the distances between target amino acid residues, was found to be a determining factor for the cross-linking efficiency. Moreover, the reactive groups of the chemical cross-linker also play an important role; a higher cross-linking reaction efficiency was found for maleimides compared to 2-pyrimidyldithiols. The reaction between maleimides and sulfhydryl groups is more favorable than that between N-hydroxysuccinimide esters and amine groups, although cysteine residues are less abundant in proteins compared to lysine residues.

Abstract 898: Probing accessibility and conformational changes in ATP-binding sites during the catalytic cycle of human P-glycoprotein (ABCB1).

Abstract P-glycoprotein (P-gp) is an ATP-binding cassette (ABC) efflux transporter implicated in the development of multidrug resistance in cancer cells. The catalytic cycle of P-gp is still not fully understood. Both the C431 and C1074 residues in the Walker A motifs of nucleotide-binding domains (NBDs) are highly conserved in P-gp from different species and these residues are excellent reporter sites to study the interaction between the two NBDs during the catalytic cycle. Using homo-bifunctional crosslinkers, disulfide crosslinking of the 431C and 1074C residues in a cysless background can be observed in the presence of copper phenanthroline (CuP) and M17M, which have spacer arm lengths of 0 Å and 25 Å, respectively. However, crosslinking with M17M was prevented in the ADP-vanadate-trapped (closed) conformation, indicating that the distance between 431C and 1074C or the accessibility of these residues changes upon ATP hydrolysis. In addition, the accessibility of the residues, separately or together (431C/1074C) in both the apo and closed conformations was determined with fluorescein-5-maleimide (FM) and methanethio-sulfonate derivatives of rhodamine (MTS-rhodamine) and verapamil (MTS-verapamil), which exhibit different physical properties. The crosslinking of cysteine residues with thiol-reactive compounds is observed both in apo (open) and ADP-vanadate-trapped (closed) conformations, though at a reduced level in the latter case, indicating the flexible nature of the cysteines in Walker A domains. Consistent with these findings, the docking of FM in a homology model of human P-gp ATP sites shows that the sulfur atoms of 431C or 1074C can be oriented either facing the ATP pocket or facing away from the pocket. A different degree of labeling was observed between the two residues; for 1074C having 1.4- fold and 2-fold higher labeling with FM compared to 431C in the apo and closed conformations respectively, suggesting that accessibility of 1074C is greater than that of 431C in both conformations. In the presence of the transport substrate cyclosporine A (50 μM), crosslinking is still observed with CuP and M17M, suggesting that the binding of substrates to the drug-binding site in the transmembrane domains does not change the accessibility of the cysteines in the NBDs. In summary, the cysteines in the Walker A motifs of human P-gp are flexible and differentially accessible in the apo and closed conformations. In addition, the NBDs of human P-gp are closer together (20-25 Å) than the NBDs observed in the X-ray structure of mouse P-gp (36 Å). Citation Format: Hong-May Sim, Jaya Bhatnagar, Samantha A. Green, Adam Gonzalez, Eduardo E. Chufan, Suresh V. Ambudkar. Probing accessibility and conformational changes in ATP-binding sites during the catalytic cycle of human P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 898. doi:10.1158/1538-7445.AM2013-898

Humic acid rejection and flux decline with negatively charged membranes of different spacer arm lengths and charge groups

A series of novel charged ultrafiltration membranes that differed in spacer arm length and charge group were generated by covalent attachment of negatively charged group to a commercially available regenerated cellulose membrane. Rejection of humic acid and flux decline were compared with essentially neutral and negatively charged versions of regenerated cellulose membranes of different spacer arm lengths and charge groups. Effects of membrane molecular weight cut-off and solution pH on humic acid removal and flux decline on neutral and negatively charged membranes of different spacer arm lengths were also compared. Results indicated that the membrane with larger spacer arm length has better rejection of humic acid and less membrane fouling compared to that with smaller spacer arm length. In addition, the modified negatively charged membrane with a strongly acidic (sulfonic acid) functional group has better rejection of humic acid and less membrane fouling than that with a weakly acidic (carboxylic acid) functional group. These experimental results are consistent with the larger charge of the membranes having larger spacer arm length and sulfonic acid group. This study confirms that the appropriate design of membrane charge functionality could be an effective way for better removal of humic acid and reduction of membrane fouling due to the electrostatic interactions with the combination effect of membrane pore size.

Negative ion fragmentations of disulfide-containing cross-linking reagents are competitive with aspartic acid side-chain-induced cleavages

It has been shown that the disulfide moiety in the chemical cross-linking reagent dithiobis(succinimidyl)propionate (DSP), which is similar in structure to the natural cystine disulfide, cleaves preferentially to the peptide backbone in the negative ion mode. However, the tandem mass (MS/MS) spectra of peptides in the negative ion mode are often dominated by products arising from low-energy, side-chain-induced processes, which may compete with any facile cross-linker fragmentations and complicate identification of chemical cross-links in a complex mixture. Two disulfide-containing crosslinking reagents similar to DSP, but with varying spacer arm lengths, were synthesized and the MS/MS spectra of several model peptides cross-linked with these reagents were investigated. Theoretical calculations were used to describe the energetics of the cross-linker fragmentations as well as several low-energy side-chain-induced fragmentations which compete with disulfide cleavages. Altering the spacer arm length of the cross-linker, such that there is one methylene group less than in DSP, results in a more facile cleavage process, whilst the opposite is true when a methylene group is added. Of the low-energy side-chain-induced fragmentations studied, only those from aspartic acid compete significantly with those of the cross-linker disulfide. Low-energy cleavage processes from aspartic acid that compete with cross-linker fragmentations occur in the negative ion MS/MS spectra of the cross-linked peptides, irrespective of the spacer arm length. Other fragmentation pathways do not significantly interfere with low-energy disulfide cleavage, making the presence of additional product ions in the MS/MS spectrum diagnostic for the presence of aspartic acid.

Peptide-functionalized chitosan–DNA nanoparticles for cellular targeting

Chitosan–pDNA nanoparticles with various weight ratios (chitosan:pDNA 1:4–8:1) were characterized for particle size, zeta potential, morphology, and pDNA binding efficiency. For targeted gene delivery applications, nanoparticles were functionalized by coupling fluorescent dye and tyrosine kinase receptor B (TrkB) binding peptides on the particle surface. The targetability of the peptide-functionalized nanoparticles was demonstrated in TrkB positive murine transformed monocyte/macrophage cells (RAW 264). It was observed that weight ratio influenced DNA condensation and nanoparticle properties. An increase in the weight ratio decreased the average particle size, but increased the zeta potential. Cell culture studies showed that TrkB-peptide-functionalized nanoparticles bound to cells more effectively than nanoparticles functionalized with a control peptide. The length of the PEG spacer arm of the amine-to-sulfhydryl crosslinker used in the functionalization was found to positively correlate with the cellular attachment efficiency. This study suggests that the peptide-functionalization could be used to target chitosan–pDNA nanoparticles to specific cells.

Cationic Spacer Arm Design Strategy for Control of Antimicrobial Activity and Conformation of Amphiphilic Methacrylate Random Copolymers

Antimicrobial and hemolytic activities of amphiphilic random copolymers were modulated by the structure of the cationic side chain spacer arms, including 2-aminoethylene, 4-aminobutylene, and 6-aminohexylene groups. Cationic amphiphilic random copolymers with ethyl methacrylate (EMA) comonomer were prepared with a range of comonomer fractions, and the library of copolymers was screened for antimicrobial and hemolytic activities. Copolymers with 4-aminobutylene cationic side chains showed an order of magnitude enhancement in their antimicrobial activity relative to those with 2-aminoethylene spacer arms, without causing adverse hemolysis. When the spacer arms were further elongated to hexylene, the copolymers displayed potent antimicrobial and hemolytic activities. The 4-aminobutylene side chain appears to be the optimal spacer arm length for maximal antimicrobial potency and minimal hemolysis, when combined with hydrophobic ethylmethacrylate in a roughly 70/30 ratio. The copolymers displayed relatively rapid bactericidal kinetics and broad-spectrum activity against a panel of Gram-positive and Gram-negative bacteria. The effect of the spacer arms on the polymer conformation in the membrane-bound state was investigated by molecular dynamics simulations. The polymer backbones adopt an extended chain conformation, parallel to the membrane surface. A facially amphiphilic conformation at the membrane surface was observed, with the primary ammonium groups localized at the lipid phoshophate region and the nonpolar side chains of EMA comonomers buried in the hydrophobic membrane environment. This study demonstrates that the antimicrobial activity and molecular conformation of amphiphilic methacrylate random copolymers can be modulated by adjustment of cationic side chain spacer arms.

Mapping Conformational Changes Associated with the Catalytic Cycle of Human P-Glycoprotein (ABCB1)

P-glycoprotein (Pgp, ABCB1) is an ABC transporter that uses the energy of ATP hydrolysis to efflux a variety of amphipathic substrates including anti-cancer agents from the cell. In this study, we have used disulfide crosslinking and Pulsed Dipolar Electron Spin Resonance Spectroscopy (PDS) to elucidate the conformational changes associated with the catalytic cycle. We generated a library of double cysteine mutants in a cys-less background and expressed in baculovirus-insect and mammalian cell expression systems. All the mutants expressed at the cell surface in HeLa cells and were also functionally active. Disulfide crosslinking was performed in crude membranes and each double cysteine mutant of Pgp was tested for its ability to crosslink with ten crosslinkers of varied spacer arm length. We observed crosslinking in four double cysteine mutants with Cys residues in TM3/TM9 (T173C/T816C), ICL2/NBD2 (A266C/I1115C), NBD1/ICL4 (A481C/S909C) and NBD1/NBD2 (A431C/A1074C). All the mutants showed modulation of crosslinking with change in protein conformation from APO to ADP-trapped. In the mutant T173C/T816C, the transmission of signal from the ATP-binding sites to TM3 and TM9 may occur via intracellular loops ICL1 and ICL3. This is highlighted by the fact that substitution of conserved residues D164 and D805 with Cys in ICL1 and ICL3, respectively reduced the basal ATPase activity and was unaffected by addition of inhibitors such as tariquidar and cyclosporine A. In addition, Cys sites in the Walker A domain of both NBD's cross linked in apo conformation to 20-25 A long crosslinkers. This suggests that the ATP sites are much closer than predicted by the homology model of human Pgp (36 A) based on the mouse Pgp structure. In aggregate, the broad distance measurements from PDS and disulfide crosslinking suggest high degree of flexibility in Pgp.

Three‐dimensionally ordered macroporous cross‐linked polystyrene incorporating functional groups via hydrophilic spacer arms: Application for heavy metal ion adsorption

AbstractThree‐dimensionally ordered macroporous cross‐linked polystyrenes (3DOM CLPS) with 2‐mercaptobenzothiazole (MBZ) groups attached directly or via hydrophilic spacer arms to frameworks were prepared for use as heavy metal ion adsorbents. The hydrophilic spacer arms are polyethylene glycol (PEG) chains with three different lengths. The functionalized 3DOM CLPS were characterized by SEM, FTIR, and elemental analysis. The results confirm that the MBZ groups have been introduced onto the pore wall of 3DOM CLPS directly or via PEGs, and the ordered structure was well preserved after functionalization. The functionalized 3DOM CLPS are effective adsorbents for the removal of heavy metal ions from solution, with adsorption capacities ranging from 0.25 to 1.14 mmol/g for silver (I) ions and 0.28 to 0.51 mmol/g for mercury (II) ions, respectively. The introduction of hydrophilic spacer arms between the 3DOM frameworks and the heterocyclic functional groups improves the adsorption capacities and adsorption kinetics. The adsorption capacities increase with the length of spacer arm. After regeneration by washing with acidic thiourea solution, the metal ion adsorption capacities of the regenerated 3DOM materials were on around 90% of the original capacities. POLYM. ENG. SCI., 2012. © 2011 Society of Plastics Engineers

Deliberate manipulation of the surface hydrophobicity of an adsorbent for an efficient purification of a giant molecule with multiple subunits

Hydrophobic interaction chromatography (HIC) is often an inevitable step for a satisfying purification in giant vaccine molecules production. But great mass and activity loss associated with poor purity often occur simultaneously. In this paper, high purity and high bioactivity recovery for the HIC process of hepatitis B surface antigen (rHBsAg) purification were achieved through manipulation of surface hydrophobicity of the adsorbent. Spacer arm length and ligand density were regulated, respectively, through which the interaction between the vaccine and the adsorbent was manipulated deliberately. It was found even in a narrow scope, varying spacer arm length and ligand density resulted in purification factor changing from 1 to 96.5, and rHBsAg recovery from 3 to 91%. The optimal purification performance was achieved when the spacer arm was C8 and the ligand density was 9.2 μmol/g suction-dried wet gel with an average distance of ligands of 3.6 nm. This deliberate regulation strategy represents a new approach of improving purification of giant multi-subunit proteins.

APN/CD13-targeting as a strategy to alter the tumor accumulation of liposomes

Targeting angiogenic vasculature has been validated as a viable approach for cancer imaging and therapy. The tumor vasculature-specific ligand asparagine–glycine–arginine (NGR) peptide targets the isoform of aminopeptidase N (APN, also referred to as CD13) that is expressed on the endothelial cells in angiogenic vessels such as the neovasculature in tumors. APN/CD13 has become widely recognized as a rational target for therapeutic development and several NGR-conjugated agents are now in pre-clinical and clinical development. In the current study, a CT image-based approach is used to evaluate the in vivo performance of several NGR conjugated liposome formulations that vary in terms of NGR density and PEG spacer arm length. Indeed, for the first time it is demonstrated that CT imaging can be used for quantitative and longitudinal assessment of the pharmacokinetics and biodistribution of an actively targeted liposome formulation containing an iodinated agent. In comparison to conventional methods, the CT image guided drug delivery approach enables visualization of the intratumoral distribution of liposomes and quantification of the fraction of tumor occupied by the vesicles over time. This study is the first to use CT for molecular imaging.

Probing fundamental film parameters of immobilized enzymes—Towards enhanced biosensor performance. Part II—Electroanalytical estimation of immobilized enzyme performance

The method of immobilization of a protein has a great influence on the overall conformation, and hence, functioning of the protein. Thus, a greater understanding of the events undergone by the protein during immobilization is key to manipulating the immobilization method to produce a strategy that influences the advantages of immobilization while minimizing their disadvantages in biosensor design. In this, the second paper of a two-part series, we have assessed the kinetic parameters of thin-film laccase monolayers, covalently attached to SAMs differing in spacer-arm length and lateral density of spacer arms. This was achieved using chronoamperometry and an electroactive product ( p-benzoquinone), which was modeled in a non-linear regressional fashion to extract the relevant parameters. Finally, comparisons between the kinetic parameters presented in this paper and the rheological parameters of laccase monolayers immobilized in the same manner (Part I of this two paper series) were performed. Improvements in the maximal enzyme-catalysed current, i max, the apparent Michaelis–Menten constant, K m and the apparent biosensor sensitivity were noted for most of the surfaces with increasing linker length. Decreasing the lateral density of the spacer-arms brought about a general improvement in these parameters, which is attributed to the decrease in multiple points of immobilization undergone by functional proteins. Finally, comparisons between rheological data and kinetics data showed that the degree of viscosity exhibited by protein films has a negative influence on attached protein layers, while enhanced protein hydration levels (assessed piezoelectrically from data obtained in Paper 1) has a positive effect on those surfaces comprising rigidly bound protein layers.

Characterization of the Oligomeric Structure of the Ca2+-activated Cl− Channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca(2+)-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl(-) secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca(2+), suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.