Surface Chemistry and Spectroscopic Study of a Cholera Toxin B

Jan 29, 2018 - Department of Engineering Design Technology, Cerritos College, 11110 Alondra Boulevard, Norwalk, California 90650, United States...
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Article Cite This: Langmuir 2018, 34, 2557−2564

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Surface Chemistry and Spectroscopic Study of a Cholera Toxin B Langmuir Monolayer Shiv K. Sharma,† Elif S. Seven,† Miodrag Micic,‡,§ Shanghao Li,‡ and Roger M. Leblanc*,† †

Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146, United States MP Biomedicals LLC, 3 Hutton Center, Santa Ana, California 92707, United States § Department of Engineering Design Technology, Cerritos College, 11110 Alondra Boulevard, Norwalk, California 90650, United States ‡

ABSTRACT: In this article, we explored the surface chemistry properties of a cholera toxin B (CTB) monolayer at the air−subphase interface and investigated the change in interfacial properties through in situ spectroscopy. The study showed that the impact of the blue shift was negligible, suggesting that the CTB molecules were minimally affected by the subphase molecules. The stability of the CTB monolayer was studied by maintaining the constant surface pressure for a long time and also by using the compression−decompression cycle experiments. The high stability of the Langmuir monolayer of CTB clearly showed that the driving force of CTB going to the amphiphilic membrane was its amphiphilic nature. In addition, no major change was detected in the various in situ spectroscopy results (such as UV−vis, fluorescence, and IR ER) of the CTB Langmuir monolayer with the increase in surface pressure. This indicates that no aggregation occurs in the Langmuir monolayer of CTB.



INTRODUCTION Cholera toxin (CT) is an oligomer protein complex of the AB5 family which consists of six subunits, secreted by bacterium Vibrio choleraes.1 It is commonly referred to as a choleragen and is often abbreviated as CTX, Ctx, or CT. It is the main virulence factor which causes an acute dehydration effect in cholera disease. Among six subunits, a single copy of cholera toxin A unit (CTA) is an enzymatically active monomer subunit consisting of two chains connected by a disulfide bond and is the toxic part of the toxin. The other five copies of cholera toxin B (CTB) form a pentamer ring, which is the nontoxic homopentamer subunit.2 (The molecular weight of each unit is 11.6 kDa.) It is also responsible for the recognition and binding to its membrane-bound receptor ganglioside GM1 followed by cell penetration.3 As the CTB subunit is nontoxic ligand to GM1 gangliosides glycans, there are many research applications of CTB. One of the very common applications of CTB is its use as a neuronal tracer when the CTB conjugated with fluorescent probes is used either as an imaging or as a quantification tool.4 Recently, CTB has been conjugated with quantum dots and nontoxic carbon dots for in vitro and in vivo imaging applications.5,6 Furthermore, as GM1 gangliosides are commonly found in the cell wall lipid rafts, the CTB is extensively used as a tool to study lipid raft morphology and distribution. Because of the ability of CTB to penetrate cells through rapid and high binding affinity to the GM1 gangliosides and is nontoxic in nature, it has been extensively used as a tool for retrograde © 2018 American Chemical Society

neuronal tracing as there are many cell surface gangliosides on the peripheral neural system.4,7 In the therapeutic world, the recombinant CTB is currently used as an add on component to the Ducoral oral cholera vaccine.8,9 In addition to being used as a component of the vaccine, CTB has many potential pharmaceutical applications, such as an immunomodulatory and anti-inflammatory agent10 and as a drug delivery system for transmucosal delivery.11,12 Furthermore, CTB can be conjugated to liposomes and modified protocells for the targeted delivery of antigens and drugs to motor neurons.13,14 CTB conjugated with saporin has been used to establish a rodent model for motor neuron death in disease.15 Because an increased number of membrane surface GM1 gangliosides are found in Alzheimer’s disease patients,16,17 CTB may be used in the future as a diagnostic tool for the detection of GM1 in amyloid plaques bound to Aβ.18 As an example of a molecule of multiple applications, its structure, function, and action mechanism of CTB are well studied.19−21 Considerable studies have also been conducted for the application and properties of CTB.22,23 However, the driving force of CTB going to the interface to bind GM1 is not clear. Although the interaction and binding affinity between GM1 and CTB has been roughly studied by various techniques (such as flow cytometry, surface plasmon resonance, and lipid Received: December 15, 2017 Revised: January 17, 2018 Published: January 29, 2018 2557

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Langmuir bilayers attached to a solid surface),24−30 it is difficult for these methods to mimic the dynamic amphiphilic membrane structure in vivo. Langmuir monolayers, a quintessential twodimensional (2-D) surface chemistry approach, can be one of the best techniques for studying these dynamic amphiphilic membranes.31,32 The structure of cell membranes comprises phospholipids bilayers, which can be divided into three regions as follows:33 (i) the interior core of the membrane, which has hydrophobic character; (ii) the hydrophilic headgroups of the lipids; and (iii) the lipid−water interfacial layer which exhibits similar dielectric permittivity to the air−water interface.34,35 Unfortunately, the surface chemistry of monolayers of CTB has not been investigated to the best of our knowledge. In addition, few publications have reported the reason that CTB forms a pentamer. Therefore, significant research about the behavior of CTB at the air−water interface must be conducted. This article presents the results of a surface chemistry study of CTB at the air−subphase interface, which can be considerably utilized to resemble the amphiphilic membranes structure in vivo. Here, we investigated the properties of CTB monolayers using the Langmuir technique as this technique is viewed as powerful in comprehending how surface proteins communicate with their membrane and subphase environment by studying their air−subphase behavior.36 Besides, this facile method has been used to reduce interfacial tension and control wetting properties by employing monolayers of chemical surfactant37 that devise the ease of restraining the intermolecular interactions and disposition of the analyte by the use of controllable variables such as the subphase, monolayer analyte, surface pressure, and surface potential. This study will make clear the amphiphilic natures of vesicles and cell membranes in cohesion with CTB that prevail in vivo and the interaction mechanism around the amphiphilic natures. More specifically, this study will be helpful in apprehending the less-wellunderstood mechanism of how bacterial toxins such as CTB cross the plasma membrane of targeted cells because, at first, these toxins bind with the lipids and proteins of cell membranes. Moreover, this approach will be beneficial to studying the property of transmembrane receptors, cell adhesion, and its implications for possible future biosensing applications and microfluidic device development.



(KSV Instrument Ltd., Helsinki, Finland) having an area of 225 cm2 (7.5 cm × 30 cm). Langmuir Monolayer Preparations. The CTB solution was prepared in pure water (pH 6.0) at a concentration of 1.78 × 10−5 M. A 20 mM citrate phosphate buffer (pH 5.5) solution was used to prepare the subphase. The CTB was spread uniformly over the air− subphase interface by using a 100 μL syringe (Hamilton Co., Reno, Nevada). The spreading volume of the toxin solution was 45 μL for the surface chemistry and spectroscopic measurements. After the solution was spread, the Langmuir monolayer was allowed to attain the equilibrium state for approximately 15−20 min. Then, the monolayer was compressed at a rate of 270 Å2·molecule−1·min−1. This compression rate was used such that the compression is complete in around 10 min to maintain the consistency in the experiments. The experiments were repeated, and good reproducibility was achieved. The reason that we employed the Kibron μ-trough for the surface pressure/potential and compression/decompression experiments and the KSV trough for in situ UV−vis and fluorescence experiments is that the KSV trough provides plenty of space to frequently relocate the UV−vis and fluorescence machine parts in comparison to the Kibron trough. UV−Vis Spectroscopy. The in situ UV−vis absorption spectra of the Langmuir monolayer were taken with the help of a model 8452 A HP spectrometer fixed on a rail close to the KSV trough (KSV Instrument Ltd., Helsinki, Finland). The KSV trough has dimensions of 7.5 cm × 30 cm, which is appropriate for reaching toward the quartz window located in the center of the trough. Fluorescence Spectroscopy. The in situ fluorescence spectra of the CTB Langmuir monolayer was measured with the help of an optical fiber detector on the top of the KSV trough, which was coupled to the Spex Fluorolog fluorespectrometer (Horiba, Jovin Yvon, Edison, NJ). The optical fiber used in the experiment had an area of 0.25 cm2 and rested approximately 1 mm above the surface of the subphase. The instrument works in such a way that the excitation light gets transmitted through the optical fiber from the light source to the monolayer and the emitted light from the monolayer gets dispatched back to the detector through the optical fiber. Furthermore, a Fluorolog-3 (slit width 5 nm) was used to measure the amount of CTB cast into the subphase at a definite time interval. Infrared External-Reflection (IR ER) Spectroscopy. A Bruker Equinox 55 FTIR instrument (Billerica, MA) equipped with the XA511 accessory for the air−water interface was used at the air−subphase interface to obtain infrared spectra. A Kibron μ-trough S (Helsinki, Finland) with dimensions of 5.9 cm × 21.1 cm was employed for the experiment. The measurements were carried out by the use of ppolarized light and a mercury−cadmium−telluride (MCT) liquidnitrogen-cooled detector. Each spectrum was acquired by the coaddition of 1200 scans at a resolution of 8 cm−1.

MATERIALS AND METHODS



Chemicals. Cholera toxin B (CTB) was obtained from SigmaAldrich with a molecular weight of 11.2 kDa that was determined using matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry. The water that was utilized in the experiments was obtained from PURELAB Ultra, Elga Lab Water (Veola Water Solutions and Technologies, U.K.) with a resistivity of 18 MΩ cm, a surface tension of 71.6 mN·m−1, and a pH of 6.0 at 20.0 ± 0.5 °C. Equipment. Every experiment was conducted in a clean room (class 1000) in which the temperature (20.0 ± 0.5 °C) and humidity (50% ± 1%) were maintained constant. In the study of surface pressure−area (π−A) isotherms, surface potential−area isotherms, stability, and compression−decompression cycles, a Kibron μ-trough (Kibron Inc., Helsinki, Finland) having area of 124.5 cm2 (5.9 cm × 21.1 cm) was used. A Kelvin probe that consists of a capacitor-like system was utilized to measure surface potential. The vibrating plate was adjusted to ∼1 mm above the surface of the Langmuir monolayer, and a gold-plated trough acted as a counter electrode. The Wilhelmy method was deployed to assess the surface pressure with a 0.51 mm diameter alloy wire probe having a sensitivity of ±0.01 mN·m−1. For the in situ UV−vis and fluorescence study, we used a KSV mini-trough

RESULTS AND DISCUSSION

Surface Pressure and Surface Potential−Area Isotherms. In order to study the interfacial behavior of CTB Langmuir monolayer under optimized conditions, the toxin was spread at the air−subphase interface. A stable monolayer was obtained by depositing 45 μL of CTB (1.7 × 10−5 M) on the subphase of a 20 mM citrate phosphate buffer that demonstrated, reproducibly, entirely essential phases of an appropriate shaping of a two-dimensional (2-D) Langmuir monolayer as shown in Figure 1. Although the toxin has a relatively low molecular weight (11.2 kDa), a solution concentration of 0.2 mg/mL was sufficient to produce a stable monolayer. From the figure, an initial zero surface pressure at 2600 Å2 molecule−1 corresponds to the gaseous phase at which the molecules at the CTB film have few to no intermolecular interactions. Starting compression via barriers resulted in an increase in surface pressure as portrayed by the decrease in area per 2558

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dimensional (3-D) liquid that continued until 580 Å2· molecule−1. The limiting molecular area which describes the minimum cross-sectional area per molecule was found to be 2200 Å2·molecule−1. This experimental value was close to the theoretical value (2350 Å2·molecule−1) obtained by a computer program for modeling, simulation, and docking: YASARA.38 To know the dipole moment or potential of the monolayer above and beneath the monolayer, a surface potential measurement was carried out simultaneously with the surface pressure measurement. This also gives an idea of molecular interactions that occur prior to and following the phase change. In the figure, there is an immediate increase in surface potential in the gaseous phase, which is due to the change in dipole moment on account of compression, indicating that surface dipole forces and the orientation of subphase molecules cause a net interfacial orientation. This further suggests that water molecules at the air−subphase interface aligned with the pendant hydrogen pointing outward together with Na+ ions from buffer outnumbered the rest of the water molecules with the negative oxygen end exposed.39 In the liquid condensed phase, the surface potential has increased, which denotes that the molecules are presumed to take an almost vertical orientation as the effective (normal) dipole moment has its utmost value. The small bumps seen in the surface potential curve are due to the movement of CTB molecules to gain a specific orientation on the subphase surface under the vibrating

Figure 1. Langmuir monolayer of CTB (0.2 mg/mL) obtained after spreading on the 20 mM citrate phosphate buffer subphase (pH 5.5).

molecule. This reveals the liquid phase of the monolayer starting at 2450 Å2·molecule−1 and ending at 900 Å2· molecule−1, where molecules are less free to move as their orientation becomes uniform. Further compression of the monolayer resulted in the cessation molecular movement known as the solid/condensed phase analogous to the three-

Figure 2. Compression−decompression Langmuir monolayer curves of CTB (0.2 mg·mL−1) at different surface pressures spread on citrate phosphate buffer (pH 5.5). The amount injected was 45 μL at room temperature. 2559

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Figure 3. Stability measurements of the CTB Langmuir monolayer at two different surface pressures, (A) 20 and (B) 30 mN·m−1, by spreading 45 μL of CTB (0.2 mg·mL−1) on the citrate phosphate buffer (pH 5.5) at room temperature.

Figure 4. UV−vis absorption spectra of the CTB Langmuir monolayer on citrate phosphate buffer (pH 5.5) (A) and a plot of absorbance versus surface pressure at wavelengths of 230 and 280 nm (B).

electrode. After attaining the solid/condensed phase at 900 Å2· molecule−1, we observed a decrease in the surface potential. This is best described as the cancellation of the dipole−dipole moment due to the short distance (surface pressure−area isotherm). From this information, it is helpful to know the role

of electrostatically charged CTB molecules in the biological process. Compression−Decompression Cycles and Stability Curves. It is essential that the monolayer should be stable long enough to perform in situ experiments. From the compression−decompression data, we determined that the 2560

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monolayer was appreciably stable. The higher peaks at 230 and 280 nm relate the increase in tyrosine and tryptophan absorbance while the CTB monolayer is achieving rigidity at higher surface pressure. As tryptophan has a higher absorbance intensity than tyrosine, in situ fluorescence spectroscopy (Figure 5) was also predominated by the tryptophan emission

CTB Langmuir monolayer can be classified as almost a reversible collapse because it prolongs the monolayer from cycle to cycle with a minimal loss of CTB molecules from the monolayer during each cycle, which can be further justified due to the presence of less hysteresis, additionally confirming the high stability of the monolayer. Three compression−decompression cycles were scrutinized at surface pressures of 20, 25, 30, and 35 mN·m−1 (Figure 2). From the figure, it was found that the hysteresis, the extent of analyte being lost to the subphase from the interface, was slightly increasing from 3.5 to 5.7% on going from 20 to 35 mN·m−1. This result justifies that the CTB molecules at the interface were not soluble in the subphase because of the rigidity of the Langmuir monolayer, and aggregates and domain formation were negligible. Since compression−decompression takes appreciable time to complete the cycles, it can also be used as a criterion to check the stability of the monolayer. To confirm the stability of the CTB Langmuir monolayer, experiments were performed at two different surface pressures, viz., 20 and 30 mN·m−1 (Figure 3). It was found that the monolayer was stable up to 28 000 s (7.78 h). This stability was achieved at a surface pressure of 20 mN·m−1. When the surface pressure was adjusted to 30 mN·m−1, the stability was suppressed. The main reason for Langmuir monolayers to be less stable at higher pressures is the inability of the monolayer to sustain the analyte in that compressed state. The high stability of the Langmuir monolayer of CTB shows that the amphiphilic nature of CTB is the driving force to push it to the amphiphilic air−subphase interface. To double check whether CTB aggregates at the air−water interface, various surface spectroscopic methods have been used to examine the Langmuir monolayer of CTB, and the results are shown below. UV−Vis Absorption Spectroscopy. After knowing the stability of the CTB Langmuir monolayer, we were interested to confirm it by perceiving the extent of analyte on the interface. Hence, the UV−vis spectra of the CTB Langmuir monolayer at different surface pressures were recorded. The absorption maxima peaks were obtained at 230, 280, and 307 nm as shown in Figure 4. The band at 230 nm can be designated as the overlapped higher-energy maxima of tyrosine and tryptophan residues obtained from the CTB molecule. The band at 280 nm can also be attributed to a combination of the absorption band of tryptophan and tyrosine residues, which are actually the external residues of the CTB molecules which remain exposed to the subphase solution. No specific assignment could be given to the peaks at 307 nm. On plotting the absorbances and surface pressures, a linear relationship was established at two different wavelengths, viz., 230 and 280 nm. The slope was approximately the same for two different plots. This observation reinforces the interpretation that the protein was retained at the interface. The increase in surface pressure leads to an increase in the absorbance at the band maximum. This is because the increasing surface pressure is due to the compression of the monolayer, which ultimately increases the number of molecules per unit area. The absorbance obtained at 0 mN·m−1 is due to the presence of the CTB Langmuir monolayer. This also highly signifies the stability of the monolayer because if the monolayer were unstable, we would not get an increase in absorbance intensity but rather would have ended at a plateau as CTB molecules are compelled to discharge into the subphase. Fluorescence of the Langmuir Monolayer. From the in situ UV−vis experiments, it was clear that the CTB Langmuir

Figure 5. In situ fluorescence spectra at the air−subphase interface of the CTB (1.7 × 10−5 M) Langmuir monolayer (A) and the plot of maximum emission at 350 nm as a function of increasing surface pressure (B).

at 350 nm.40 The figure shows that the dominance of tryptophan emission was achieved from the gaseous phase to the solid/condensed phase, which actually corresponds to the observation achieved from absorbance data at similar surface pressures. Furthermore, we were surprised to see the negligible impact of the blue shift as seen in DPPC41 and β-galactosidase.42 A blue shift occurs by altering the absorbance maxima and emission maxima, but we achieved the absorbance and emission at 280 and 350 nm, respectively, which suggests that the CTB molecules were minimally affected by the subphase molecules. We were also concerned about the possibility of loss of analyte from the monolayer after the completion of maximum compression. Therefore, we measured the fluorescence intensity of the solution directly withdrawn from the subphase after 2 h of wait time in 15 min intervals up to 3 h (Figure 6). We observed that there was a slight decrease in intensity which might be associated with (i) the solubilization of protein, (ii) the aggregation of protein, and (iii) the impact of wait time on 2561

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Figure 6. Plot of fluorescence intensity vs wait time of the CTB Langmuir monolayer.

the intensity of light. From the stability curve, we can outstrip the first point. On performing UV−vis at the air/subphase interface for the CTB monolayer, the increase in absorption maxima after several rounds of experiment also justifies our hypothesis that the decrease in intensity is not due to the solubilization of protein. The observed decrease in intensity could be due to the slight aggregation of CTB at the monolayer or due to the impact of wait time on the intensity of light. Infrared External-Reflection (IR ER) Technique. After having observed that there was slight decrease in intensity while performing fluorescence spectroscopy (of the solution) obtained by withdrawing a small amount of subphase from the trough, we suspected that it might be due to the modest aggregation of CTB molecules. To confirm this, we performed an IR ER experiment. The surface selection rule for the IR ER measurement technique is that when the angle of incidence is higher than Brewster’s angle of the substrate, the surface parallel (p) component of a transition moment yields a positive peak and vice versa.43 Brewster’s angle for the air−water interface is 53°, and the air subphase that we have used should have a lower Brewster’s angle (approximately 50°). Figure 7a shows the normalized p-polarized IR ER spectra of the CTB Langmuir monolayer at a surface pressure of 15 mN·m−1 with different incident angles, in accordance with the surface selection rule. Figure 7b,c exhibits the p-polarized IR ER spectra of CTB for different surface pressures at an incident angle of 60°. We chose this angle because it had a high signalto-noise ratio. While examining the amide I region (1700−1600 cm−1), amide II region (1600−1500 cm−1), and even amide III region (1400−1300 cm−1), we found that none of the bands were shifting with increasing surface pressure. The peak at 1651 cm−1 resembles the α-helix (amide I) predominantly due to the CO (symmetric) and C−N stretching of the amide group whereas the peaks at 1627 and 1685 cm−1 are due to the antiparallel β-sheet.44 The peak at 1700 cm−1 specifies the βturn and the antiparallel β-sheet basically due to the absorption from the turn and transition dipole coupling in the antiparallel β-sheet. The peak observed at 1545 cm−1 is due to the α-helix (amide II) which arises from the N−H bending (60%) and C−N stretching (40%). The C−H stretching peaks are found in the range of 2800−3000 cm−1, and C−O peaks are observed at around 1200 and 1300 cm−1 whereas the peak at 1720 cm−1 is

Figure 7. Normalized p-polarized IR ER spectra of the CTB Langmuir monolayer (a) at a surface pressure of 15 mN·m−1 with different incident angles, (b) for a comparison of spectra for different surface pressures at an incident angle of 60°, and (c) expanded spectra to show the amide I and II regions clearly.

due to the CO stretching vibration.45 The peaks from 3400 to 3250 cm−1 resemble the N−H stretch due to primary and secondary amines. The peak at 2357 cm−1 is due to CO2, and typically this is significant if the air-subphase is not covered or if covered there is not sufficient nitrogen flow to reduce the CO2 2562

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Langmuir peak. The peak at 1456 cm−1 could not be assigned. As expected, the band positions in the amide I region exhibited the dominancy of the β-sheet in the secondary structure of CTB due to the crystalline structure of the molecule.21 Having seen that no bands are changing appreciably, it was evident that there was no denaturation or aggregation of the CTB molecules on the subphase of citrate phosphate buffer. Furthermore, the intensities of bands remaining almost constant at various surface pressures indicate that the amide chains are oriented parallel to the air−subphase interface. Hence, it confirms that the slight decrease in fluorescence intensity was due to the wait time impact; i.e., the intensity of the fluorescence light may decrease during the experiment, which results in a decrease in fluorescence intensity.

(2) Leitch, J. J.; Brosseau, C. L.; Roscoe, S. G.; Bessonov, K.; Dutcher, J. R.; Lipkowski, J. Electrochemical and PM-IRRAS characterization of cholera toxin binding at a model biological membrane. Langmuir 2013, 29, 965−976. (3) Lopez, P. H.; Schnaar, R. L. Gangliosides in cell recognition and membrane protein regulation. Curr. Opin. Struct. Biol. 2009, 19, 549− 557. (4) Conte, W. L.; Kamishina, H.; Reep, R. L. Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats. Nat. Protoc. 2009, 4, 1157−1166. (5) Chakraborty, S. K.; Fitzpatrick, J. A.; Phillippi, J. A.; Andreko, S.; Waggoner, A. S.; Bruchez, M. P.; Ballou, B. Cholera toxin B conjugated quantum dots for live cell labeling. Nano Lett. 2007, 7, 2618−2626. (6) Zhou, N.; Hao, Z.; Zhao, X.; Maharjan, S.; Zhu, S.; Song, Y.; Yang, B.; Lu, L. A novel fluorescent retrograde neural tracer: cholera toxin B conjugated carbon dots. Nanoscale 2015, 7, 15635−15642. (7) Lanciego, J. L.; Wouterlood, F. G. A half century of experimental neuroanatomical tracing. J. Chem. Neuroanat. 2011, 42, 157−183. (8) Sanchez, J.; Holmgren, J. Recombinant system for overexpression of cholera toxin B subunit in Vibrio cholerae as a basis for vaccine development. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 481−485. (9) Sanchez, J.; Johansson, S.; Löwenadler, B.; Svennerholm, A.; Holmgren, J. Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination. Res. Microbiol. 1990, 141, 971−979. (10) Zhang, B.; Li, H.; Zhang, L.; Tan, S.; Cai, W.; Men, X.; Huang, Y.; Shan, Y.; Lin, Y.; Lu, Z. Anti-inflammatory effect of cholera toxin B subunit in experimental stroke. J. Neuroinflammation 2016, 13, 1−14. (11) Sun, J.-B.; Holmgren, J.; Czerkinsky, C. Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 10795−10799. (12) Holmgren, J.; Adamsson, J.; Anjuère, F.; Clemens, J.; Czerkinsky, C.; Eriksson, K.; Flach, C.-F.; George-Chandy, A.; Harandi, A. M.; Lebens, M. Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA. Immunol. Lett. 2005, 97, 181−188. (13) Harokopakis, E.; Childers, N. K.; Michalek, S. M.; Zhang, S. S.; Tomasi, M. Conjugation of cholera toxin or its B subunit to liposomes for targeted delivery of antigens. J. Immunol. Methods 1995, 185, 31− 42. (14) Porras, M. A. G.; Durfee, P. N.; Gregory, A. M.; Sieck, G. C.; Brinker, C. J.; Mantilla, C. B. A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells. J. Neurosci. Methods 2016, 273, 160−174. (15) Nichols, N. L.; Vinit, S.; Bauernschmidt, L.; Mitchell, G. S. Respiratory function after selective respiratory motor neuron death from intrapleural CTB−saporin injections. Exp. Neurol. 2015, 267, 18−29. (16) Matsuzaki, K.; Kato, K.; Yanagisawa, K. Aβ polymerization through interaction with membrane gangliosides. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2010, 1801, 868−877. (17) Yanagisawa, K. Role of gangliosides in Alzheimer’s disease. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 1943−1951. (18) Thakur, G.; Micic, M.; Leblanc, R. M. Surface chemistry of Alzheimer’s disease: a Langmuir monolayer approach. Colloids Surf., B 2009, 74, 436−456. (19) Craig, L.; Taylor, R. K. The vibrio cholerae toxin coregulated pilus: structure, assembly and function with implications for vaccine design. Bacterial Pili: Structure, Synthesis and Role in Disease; CABI: Wallingford, 2014; p 27, doi: 10.1079/9781780642550.0001. (20) Day, C. A.; Kenworthy, A. K. Functions of cholera toxin Bsubunit as a raft cross-linker. Essays Biochem. 2015, 57, 135−145. (21) Zhang, R.-G.; Scott, D. L.; Westbrook, M. L.; Nance, S.; Spangler, B. D.; Shipley, G. G.; Westbrook, E. M. The threedimensional crystal structure of cholera toxin. J. Mol. Biol. 1995, 251, 563−573. (22) Branson, T. R.; McAllister, T. E.; Garcia-Hartjes, J.; Fascione, M. A.; Ross, J. F.; Warriner, S. L.; Wennekes, T.; Zuilhof, H.; Turnbull, W.



CONCLUSIONS We found that CTB reproducibly forms a highly stable Langmuir monolayer. We also showed that there was an insignificant impact of the blue-shift during the in situ spectroscopic studies of the CTB molecule, which suggests that the analyte was not affected by subphase molecules at all. Besides this, CTB remained active on the monolayer, and no aggregation was observed, which fits this molecule in diverse pharmaceutical applications because the exploration of biological reactions at surfaces is necessary after the development of biomaterials for medicals implants such as blood-serum contact devices and intraocular instruments. In this perspective, the biocompatibility of an analyte is directly related to the surface properties of the substance. For instance, whenever a toxin meets the blood, the rapid adsorption of proteins occurs on its surface, which is known as nonspecific protein adsorption. This adsorbed protein on the surface determines the platelets’ adhesion, which enacts a prime capacity in thrombogenesis, and to prevent this, various surface modifications should be performed. Hence, the investigation of the CTB surface property will help in identifying myriad surface modification techniques. Moreover, it makes it easier to assimilate the surface chemistry behavior of this benign toxin in developing a biosensor and microfluidic devices, studying cell adhesion, understanding the property of transmembrane receptors, and acting as a model system vaguely representing cell membrane environments. On the basis of the results of surface chemistry and spectroscopic studies in this article, the lipid−subphase interfacial layer which is similar to the air− water interface is indicated to play an important role in driving the CTB to the cell membrane. On the other hand, the role of the lipid−subphase interfacial layer in the aggregation of CTB to form a pentamer ring can be ruled out.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone +1-305-284-2194. Fax: +1305-284-6367. ORCID

Roger M. Leblanc: 0000-0001-8836-8042 Notes

The authors declare no competing financial interest.



REFERENCES

(1) Holmgren, J. Actions of cholera toxin and the prevention and treatment of cholera. Nature 1981, 292, 413−417. 2563

DOI: 10.1021/acs.langmuir.7b04252 Langmuir 2018, 34, 2557−2564

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DOI: 10.1021/acs.langmuir.7b04252 Langmuir 2018, 34, 2557−2564