Heterobifunctional Cross-linker Research Articles

Preparation of Immunotoxin Herceptin-Botulinum and Killing Effects on Two Breast Cancer Cell Lines

Worldwide, breast cancer is the most common cancer diagnosed among women and a leading cause of cancer deaths. The age of onset in Iran has become reduced by a decade for unknown reasons. Herceptin, a humanized monoclonal antibody, is a target therapy for breast cancer cells with over expression of HER2- neu receptors, but it is an expensive drug with only 20% beneficial rate of survival. This study introduces a novel approach to enhance the efficacy of this drug through immunoconjugation of the antibody to botulinum toxin. Decreasing the cost and adverse effects of the antibody were secondary goals of this study. Botulinum toxin was conjugated with Herceptin using heterobifunctional cross linkers, succinimidyl acetylthiopropionate (SATP) and sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) according to the supplier's guidelines and tested on two breast cancer cell lines: SK-BR-3 and BT-474. Toxin and Herceptin were also used separately as controls. The cytotoxicity assay was also performed using the new bioconjugate on cultured cells with Alamar blue and a fluorescence plate reader. Herceptin-Toxin bioconjugation significantly improved Herceptin efficacy on both breast cancer cell lines when compared to the control group. Toxin-Herceptin bioconjugation can be a potential candidate with increased efficiency for treating breast cancer patients with over expression of the HER2 receptor.

Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.

An immunoassay for the detection of triclosan-O-glucuronide, a primary human urinary metabolite of triclosan.

Triclosan-O-glucuronide (TCSG) is one of the primary urinary metabolites of the antibacterial compound triclosan or TCS that is found in many personal care products and consumer goods. We have developed a competitive, indirect heterologous ELISA for the detection of the target TCSG in urine. Such an ELISA for TCSG could be developed as a useful tool to measure this important biomarker of human exposure to TCS. Immunogens were prepared by conjugating TCSG to thyroglobulin, via heterobifunctional cross-linkers AEDP or 3-[(2-aminoethyl)dithio] propionic acid•hydrochloride and TFCS or N-[ε-trifluoroacetylcaproyloxy]succinimide ester. The coating antigen was prepared by the direct conjugation of TCSG to bovine serum albumin. Antibodies raised in rabbits 2619, 2621 (immunogen TCSG-AEDP-Thy), and 2623 (immunogen TCSG-TFCS-Thy), and the coating antigen were screened and characterized to determine their optimal concentrations. The optimized ELISA, developed with antibody 2621, gave an IC50 value of 2.85ng/mL, with the linear range (IC20-IC80) determined to be 2.6-24.8ng/mL. Selectivity of the assay was assessed by measuring cross-reactivity of antibody 2621 to related congeners such as the aglycone TCS, triclosan-O-sulfate, triclocarban, a polybrominated diphenyl ether derivative, and 3-phenoxybenzyl alcohol glucuronide. There was virtually no recognition by antibody 2621 to any of these cross-reactants. Graphical Abstract Urinary biomarker analysis of triclosan glucuronide.

Abstract P1-01-13: Triple negative breast cancer targeting paramagnetic nanoparticle for non-invasive tumor imaging

Abstract Triple-negative breast cancer (TNBC) refers to a subtype of breast cancer that is negative for estrogen receptors (ER) and/or progesterone receptors (PR) , and lacks HER2 overexpression. Therefore, this subtype of breast cancer lacks the benefits of specific therapies which target these receptors. TNBC has been characterized by an acidic extracellular environment. In human aggressive breast tumors, pHe has been measured by microelectrode and the values are in good agreement with the values observed in animal systems i.e. that the pHe is significantly acidic in the range from 6.2 to 7.0. However, tumor intracellular pH is either neutral or alkaline. Interestingly, a similar pH gradient is not observed in normal tissues. Therefore, this acidic extracellular pH (pHe) within tumor tissues can be exploited for targeted delivery of drugs and imaging agents. Recently, A pH low insertion peptide (pHLIP) derived from the protein bacteriorhodopsin has been found to target tumor acidic pH. The peptide inserts across cell membranes as an α-helix when extracellular pH (pHe) is acidic, but does not form the helix at normal or alkaline pH. Since aggressive TNBC has an acidic environment, the pHLIP will insert into the cancer cell membrane, but the pHLIP will not insert into the cell membranes of normal tissues, providing excellent specificity for targeting TNBC. Here, we demonstrate that pHLIP-tagged nanoparticles bind to and are internalized by TNBC cells in vitro. Systemic delivery of the Gd-G5-pHLIP leads to accumulation of the nanoparticles in a flank mouse model of TNBC tumor that are detected by optical and MR imaging. We have synthesized pH-responsive MRI nanoprobe, phosphonate G5-(GdDOTA-4AmP) by following our published synthetic method. The MW of the conjugated G5 dendrimer was estimated at 79,082 g/mole by maldi-tof analysis. This corresponds to a G5-dendrime with an average of 44 chelated Gd3+ ions per dendrimer. Gd44-G5 dendrimer was reacted with heterobifunctional cross-linker (sulfo-SMCC) to form reactive maleimides and then maleimide- Gd44-G5 dendrimer was coupled with C-terminus cysteine group of biotinylated Bt-pHLIP (AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG-pegBiotin). The HABA assay with biotin and avidin revealed that on average 3.1 molecules of biotin are present in Gd44-G5-Bt-pHLIP dendrimer. Finally, Rhodamine dye was conjugated to amines surface of preloaded Gd44-G5-Bt-pHLIP3 in order to achieve final conjugate Rhodamine-Gd44-G5-Bt-pHLIP3. To study pH-dependent translocation of molecules across the cell membrane, we have added Rho-Gd-G5-Bt-pHLIP to the cells and incubated for 3 h at pH 7.4 and 6.5. The cellular uptake of Rho-Gd-G5-pHLIP was significantly higher at pH 6.5. When Rho-Gd-G5 was used, the cellular uptake was considerable lower at both, pH 6.5 and 7.4. Hence, we have shown the ability of pHLIP peptide for intracellular delivery of Gd-G5 nanoparticles in vitro at pH 6.5 but the same ability is attenuated significantly at neutral pH. We have created a mouse model of TNBC MDA-MB-231 tumor. The pharmacokinetics of Gd44-G5-pHLIP was visualized in the MDA-MB-231 tumor over the course of 105 min post-contrast administration. Citation Format: Meser M Ali, Nadimpalli RS Varma, Branislava Janic, Li Zhang, James R Ewing. Triple negative breast cancer targeting paramagnetic nanoparticle for non-invasive tumor imaging [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-01-13.

Influence of Chemical Conjugation Strategies on Fibronectin's Bioactivity

Since thirty years, efforts were put forward to engineer materials that could be used to build up biomedical devices that have to come in contact with human tissues. The first devices that were developed were designed to reproduce the basic functions of the tissue or organ that they were intended to replace. For example, the first designs of arterial prostheses were made from materials that can be formed as tubes with sufficient mechanical properties and chemical stability, to withstand the pulsatile blood pressure for many years without failure. Only few concerns were raised regarding the interaction between the material and the physiological environment. Nowadays, the common strategy consists in developing materials that are likely to proactively interact with their environment.For instance, one idea is to coat the surface of biomaterials with proteins of the extracellular matrix, therefore promoting cell adhesion and proliferation. Fibronectin is one of these proteins and has been the subject of several investigations as to its potential to be used to promote cell/material interaction once conjugated to the surface of biomaterials. In this context, the aim of this study was to compare two strategies of FN immobilization in regards to the amount of bound FN and its biological activity. Two heterobifunctional cross-linkers were used to conjugate FN to ammonia plasma-treated PTFE: glutaric anhydride (GA) and Sulfo-succinimidyl 4-[p-maleimidophenyl] butyrate (sulfo-SMPB). In addition, we have also investigated the influence of adding either a hydrophilic or hydrophobic spacer between the protein and the surface on the protein bioactivity. On one hand, our results demonstrated that fibronectin RGD sequences are more available when the protein is conjugated through its lysine moieties. On the other hand, an hydrophobic spacer between fibronectin and the biomaterial surface is also likely to promote the protein bioactivity.

Infusion of Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate-Conjugated MOG35-55-Coupled Spleen Cells Effectively Prevents and Reverses Experimental Autoimmune Encephalomyelitis in Mice.

In this study, we have evaluated our recently developed method for antigen-cell coupling using sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) heterobifunctional crosslinker in prevention and reversal of experimental autoimmune encephalomyelitis (EAE). We demonstrate that infusion of MOG35–55-coupled spleen cells (MOG-SP) significantly prevents and reverses EAE. Further studies show that the protected animals exhibit significantly delayed EAE upon EAE reinduction. Moreover, adoptive transfer of CD4+ T cells from the protected mice to naïve syngeneic mice renders the recipient mice resistant to EAE induction. Unexpectedly, CD4+ T cell proliferation is similar upon ex vivo stimulation by MOG35–55 amongst all groups. However, further analysis of those proliferating CD4+ T cells shows remarkable differences in Foxp3+ regulatory T cells (70% in MOG-SP groups versus 10–25% in control groups) and in IL-17+ cells (2-3% in MOG-SP groups versus 6–9% in control groups). In addition, we discover that MOG-SP treatment also significantly attenuates MOG35–55-responding IFN-γ-producing Th1 cells. These findings suggest that MOG-SP treatment induces EAE protective MOG35–55-specific regulatory T cells and suppresses EAE pathogenic Th17 and Th1 cells. Our study provides a novel approach for antigen-based EAE immunotherapy, which can potentially be translated into clinical application for immunotherapy of multiple sclerosis.

Decreasing the Humoral Response to Factor VIII By Targeted Deletion of Factor VIII - Specific B Cells

The development of neutralizing anti-factor VIII (fVIII) antibodies (inhibitors) remains the most significant complication in the treatment of hemophilia A patients. Treatment of inhibitor patients consists of management of bleeding episodes using bypassing agents or porcine fVIII. Inhibitors can be eradicated by immune tolerance induction (ITI) using thrice-weekly administration of large doses of fVIII. However, ITI fails in approximately 30% of patients. Additionally, the median time to tolerance in successful cases is ~18 months, making ITI expensive and inconvenient. In the current study, we used a murine E16 hemophilia A model to test a novel approach to both prevent and eradicate fVIII inhibitors. We hypothesized that conjugation of fVIII to the toxin saporin, a Type I ribosome-inactivating protein, would target fVIII-specific cell surface immunoglobulin and selectively delete fVIII-specific naïve and memory B cells. Recombinant full-length fVIII was covalently linked to saporin using the heterobifunctional crosslinker N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). To test for eradication of existing fVIII inhibitors by fVIII-saporin, an adoptive transfer protocol was developed to measure fVIII-specific memory B cells. Hemophilia A donor mice were immunized with 2 μg of full-length fVIII by intravenous injection every other week for 8 weeks, followed by a final dose of 4 μg at ten weeks. Four weeks later, the mice were randomized into three treatment groups to receive equimolar doses of saporin, fVIII, or fVIII-saporin. Seven days after treatment, the mice were sacrificed and 4 x 106 plasma cell CD138+-depleted splenocytes were adoptively transferred as a source of fVIII-specific memory B cells into naïve recipient hemophilia A mice. At 24 hours, recipient mice were given a single injection of 0.5, 1.0 or 2.0 μg of recombinant full-length fVIII by tail vein injection. Anti-fVIII IgG antibodies in recipient mice were measured by ELISA 2 and 5 weeks following the fVIII injection. In the absence of fVIII-specific memory B cells from donor mice, naïve hemophilia A mice did not produce detectable anti-fVIII antibodies. Recipient hemophilia A mice receiving splenocytes from fVIII donor and saporin donor mice displayed a dose-dependent increase in anti-fVIII antibodies. In contrast, the slope of the anti-fVIII titer versus dose of fVIII was significantly decreased in recipient mice receiving splenocytes from fVIII-saporin donor mice. To test for prevention of fVIII inhibitor formation by fVIII-saporin, naïve hemophilia A mice were divided into three treatment groups to receive a single dose of saporin, fVIII, or fVIII-saporin by tail vein injection. Seven days after treatment, the mice were immunized by tail vein injection with 2 μg of full-length fVIII every other week for 10 weeks. Anti-fVIII IgG antibodies were measured 1 week after the fourth and sixth injections of fVIII. Anti-fVIII antibody titers were significantly lower in the fVIII-saporin group compared to the fVIII group (1,900 vs. 21,400 (p=0.027, n=4, Mann-Whitney test, see figure) after the fourth injection. After 6 injections, the average anti-fVIII titer of the fVIII group was 23,000 compared to 4,000 in the fVIII-saporin group (p=0.057, n=4, Mann-Whitney test, see figure). In conclusion, our results suggest that infusion of fVIII-saporin results in the depletion of both fVIII-specific naïve B cells and memory B cells. FVIII-saporin potentially could be used in the treatment of congenital hemophilia A patients with inhibitors and patients with acquired hemophilia A. In addition, fVIII-saporin potentially could be used in previously untreated patients with hemophilia A to prevent inhibitor development. Similar therapeutic strategies could be extended to other antigen-specific immune disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeted Combinatorial Therapy Using Gold Nanostars as Theranostic Platforms

This paper reports the development of a multimodal therapy nanoplatform based on gold nanostars (Au NS) as core particles. These NS were functionalized with the chemotherapeutic drug doxorubicin (DOXO), which was conjugated to the NS surface by means of a cleavable heterobifunctional cross-linker (sulfo-LC-SPDP) to allow its release under the action of reducing enzymes. To ensure a specific delivery of the chemotherapeutic drug, the nanoplatform was additionally functionalized with folic acid (FA) as targeting ligand and cellular uptake adjuvant. By synthetically modifying the plasmon band of Au NS to the near-infrared (NIR) region of the electromagnetic spectrum, the present nanoplatform was able to simultaneously combine the capability of photothermal therapy (PTT) through the conversion of absorbed light energy into localized heat and chemotherapy, enabling their monitoring by means of optical fluorescence imaging thanks to DOXO’s autofluorescence. Cellular uptake was observed to be enhanced when the A...

Isotopically-coded short-range hetero-bifunctional photo-reactive crosslinkers for studying protein structure.

Structural proteomics can be used for studying protein structures which may be difficult to examine by traditional structural biology methods such as NMR or X-ray crystallography. Crosslinking in particular is used to provide distance constraints for molecular modeling of individual proteins and protein complexes. The shortest distance constraints are most valuable for the modeling process. To be able to provide such short distance constraints, non-specific photo-reactive chemistry can be used for crosslinking reactions. However, detection of such non-specific crosslinks is difficult because the signal from any particular crosslink is low due to the broad reactivity of the crosslinking reagents. To overcome this problem, we have employed isotopic labeling of these crosslinkers. In this paper, we have demonstrated their effectiveness for studying the native α-synuclein protein structure. The non-specific reactivity, in combination with isotopic coding of these crosslinkers, allowed for the formation and detection of short-range crosslinks, targeting a variety of amino acids. These reagents may prove useful for future applications to a variety of protein structural problems. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Novel cleavable cell-penetrating peptide-drug conjugates: synthesis and characterization

We report the first drug conjugate with a negatively charged amphipathic cell-penetrating peptide. Furthermore, we compare two different doxorubicin cell-penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline-rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N-terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C-13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug-release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF-7 and HT-29 cell lines.

Methods for Identification of State-Dependent Crosslinks for Structural Determination of Membrane Proteins

Determining the structure of membrane proteins is critical to understanding how they function. Though various techniques exist to elucidate structural information, often times they lack the ability to determine allosteric movements associated with the dynamic nature of the proteins. Development of a method that affords the possibility of mapping changes in structure associated with the complicated allosteric mechanisms of membrane proteins is a valuable tool to better understanding the structural basis of protein function. The glycine receptor (GlyR) is a ligand-gated ion channel associated with inhibition of signal propagation in the central nervous system. Single-Cys mutant homomeric human α1 GlyR is overexpressed in insect cells, purified, and reacted with a methanethiosulfonate-benzophenone heterobifunctional crosslinker containing an alkyne tag. After state-dependent photoactivation in the presence or absence of modulatory ligands, inter- and intra-subunit crosslinks may be isolated by proteolysis, reduction, click chemistry and affinity chromatography. Verification and quantitation of crosslinked species may be sensitively identified by fluorescent tagging and use of a HPLC-microfluidic-laser induced fluorescence (LIF) system. Structural information can be derived through the implementation of multi-dimensional mass spectrometry to identify sites of crosslinking and relative distances which can be used to refine computational models in state specific manner. Development of this method, using GlyR as a model system, will allow for structural determinations to be made on any protein and thus allow for a better understanding of the dynamic nature of allosteric proteins.

Selective, Bead-Based Global Peptide Capture Using a Bifunctional Cross-Linker

Peptides are important species for a variety of biological functions. Detection and analysis of these molecules can be complicated by the presence of background matrix or contaminants. Therefore, a selective method to capture peptides could provide researchers with an option to isolate these remarkable species. Our goal was to perform a set of experiments that would validate the concept of a novel, selective peptide capture, whereby peptides are isolated on functionalized magnetic beads through the use of the heterobifunctional cross-linker, Sulfo-LC-SPDP. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to monitor mass changes associated with the cross-linking reaction. MALDI-TOF MS was then used to monitor conjugation between the cross-linked peptides and sulfhydryl magnetic beads by analyzing supernatant solutions for the presence or absence of cross-linked peptide. Through these experiments, we have proof of concept data confirming that peptides can be isolated on sulfhydryl magnetic beads by using Sulfo-LC-SPDP. This method is a suitable selective global peptide isolation strategy to separate the molecules from contaminating species or sample matrix. This novel method has a variety of potential applications and detection methods.

Graphene-based immunoassay for human lipocalin-2

We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4°C in 0.1M phosphate-buffered saline (PBS) for 8weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings.

Immunization against active ghrelin using virus-like particles for obesity treatment.

Ghrelin is a gut hormone that stimulates food intake. In physiological conditions, ghrelin plasma levels rise with fasting and decrease after meals. Obese individuals have low fasting ghrelin levels that rise after food restriction, which is pointed out as a reason for the difficulty in maintaining weight loss. Some bariatric surgery procedures prevent rise in ghrelin levels with weight loss and this has been hypothesised to contribute to the long-term success of the treatment. The main goal of this study was to develop a safe and effective anti-ghrelin vaccine for obesity, through the chemical conjugation of ghrelin with a virus like particle, namely NS1 protein tubules from the Bluetongue Virus (BTV) using a hetero-bifunctional cross linker. Male adult C57BL/6 mice, with a normal weight and with diet-induced obesity (DIO), were randomized into six weight matched groups (n=6/group) and each group of mice received three intra-peritoneal injections with two weeks intervals, containing either 75 µg of ghrelin-NS1 immunoconjugate, 75 µg of NS1 or PBS. Our data show that immunized animals present increasing titres of anti-ghrelin antibodies, while their cumulative food intake significantly decreased and energy expenditure was significantly enhanced, although there were no significative changes in body weight.Vaccinated DIO mice also displayed significant decrease of NPY gene expression in the basal hypothalamus reflecting a decrease in central orexigenic signals. This study suggests that this anti-ghrelin vaccine has a positive impact on energy homeostasis and may be an additional therapeutical tool to be used with diet and exercise for obesity treatment.

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.

Bioactive albumin functionalized polylactic acid membranes for improved biocompatibility

Biocompatibility is a major challenge for successful application of many biomaterials. In this study the ability to coat chemically and enzymatically activated poly(L-lactic acid) (PLA) membranes with heat denatured human serum albumin to improve biocompatibility was investigated. PLA membranes hydrolyzed with NaOH or cutinase and then treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDAC) as a heterobifunctional cross-linker promoted the coupling COOH groups on PLA membranes and NH2 groups of heat denatured human serum albumin. This resulted in increased hydrophilicity (lowest water contact angles of 43° and 35°) and highest antioxidant activity (quenching of 79μM and 115μM tetramethylazobisquinone (TMAMQ) for NaOH and cutinase pretreated membranes, respectively). FTIR analysis of modified PLA membranes showed new peaks attributed to human serum albumin (amide bond, NH2 and side chain stretching) appearing within 3600–3000cm−1 and 1700–1500cm−1 (Fig. 3). MTT studies also showed that osteoblasts-like and MC-3T3-E1 cells viability increased 2.4 times as compared to untreated PLA membranes. The study therefore shows that this strategy of modifying the surfaces of PLA polymers could significantly improve biocompatibility.

Conjugation of glucosamine with Gd3+-based nanoporous silica using a heterobifunctional ANB-NOS crosslinker for imaging of cancer cells.

BackgroundThe aim of this study was to synthesize Gd3+-based silica nanoparticles that conjugate easily with glucosamine and to investigate their use as a nanoprobe for detection of human fibrosarcoma cells.MethodsBased on the structure of the 2-fluoro-2-deoxy-D-glucose molecule (18FDG), a new compound consisting of D-glucose (1.1 nm) was conjugated with a Gd3+-based mesoporous silica nanoparticle using an N-5-azido-2-nitrobenzoyloxy succinimide (ANB-NOS) crosslinker The contrast agent obtained was characterized using a variety of methods, including Fourier transform infrared spectroscopy, nitrogen physisorption, thermogravimetric analysis, scanning and transmission electron microscopy, and inductively coupled plasma atomic emission spectrometry (ICP-AES). In vitro studies included cell toxicity, apoptosis, tumor necrosis factor-alpha, and hexokinase assays, and in vivo tests consisted of evaluation of blood glucose levels using the contrast compound and tumor imaging. The cellular uptake study was validated using ICP-AES. Magnetic resonance relaxivity of the contrast agent was determined using a 1.5 Tesla scanner.ResultsANB-NOS was found to be the preferred linker for attaching glucosamine onto the surface of the mesoporous silica nanospheres. The r1 relaxivity for the nanoparticles was 17.70 mM−1s−1 per Gd3+ ion, which is 4.4 times larger than that for Magnevist® (r1 approximately 4 mM−1s−1 per Gd3+ ion). The compound showed suitable cellular uptake (75.6% ± 2.01%) without any appreciable cytotoxicity.ConclusionOur results suggest that covalently attaching glucosamine molecules to mesoporous silica nanoparticles enables effective targeted delivery of a contrast agent.

Power generation from a hybrid biological fuel cell in seawater

A hybrid biological fuel cell (HBFC) comprised of a microbial anode for lactate oxidation and an enzymatic cathode for oxygen reduction was constructed and then tested in a marine environment. Shewanella oneidensis DSP-10 was cultivated in laboratory medium and then fixed on a carbon felt electrode via a silica sol–gel process in order to catalyze anodic fuel cell processes. The cathode electrocatalyst was composed of bilirubin oxidase, fixed to a carbon nanotube electrode using a heterobifunctional cross linker, and then stabilized with a silica sol–gel coating. The anode and cathode half-cells provided operating potentials of −0.44 and 0.48V, respectively (vs. Ag/AgCl). The HBFC maintained a reproducible open circuit voltage >0.7V for 9d in laboratory settings and sustained electrocatalytic activity for >24h in open environment tests.

A Simple Bioconjugate Attachment Protocol for Use in Single Molecule Force Spectroscopy Experiments Based on Mixed Self-Assembled Monolayers

Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin.

Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm−1). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.